Use of methylsulfonylmethane (msm) to modulate microbial activity

ABSTRACT

Disclosed herein are methods of use of methylsulfonylmethane (MSM) to modulate microbial activity, such as to enhance or inhibit the activity of microorganisms. In one example, MSM (such as about 0.5% to 5% MSM) is used to enhance fermentation efficiency, such as to enhance fermentation efficiency associated with the production of beer, cider, wine, a biofuel, dairy product or any combination thereof. Also disclosed are in vitro methods for enhancing the growth of one or more probiotic microorganisms and methods of enhancing growth of a microorganism in a diagnostic test sample. Methods of inhibiting microbial activity are also disclosed. In one particular example, a method of inhibiting microbial activity includes selecting a medium that is susceptible to H1N1 influenza contamination; and contacting the medium with MSM at a concentration of about 10% to about 16% of weight by volume, thereby inhibiting H1N1 influenza microbial activity.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to PCT/US2010/054845, filed Oct. 29,2010; U.S. Provisional Applications No. 61/257,751 filed Nov. 3, 2009;No. 61/259,098 filed Nov. 6, 2009; No. 61/294,437 filed Jan. 12, 2010;and No. 61/256,935, filed Oct. 30, 2009; each of which is herebyincorporated by reference in its entirety.

FIELD OF THE DISCLOSURE

This disclosure relates to the field of methylsulfonylmethane (MSM),specifically to methods of use of MSM to modify biological activity,such as to enhance or inhibit microbial activity including bacterialgrowth.

BACKGROUND

Microorganisms (or microbes) are microscopic organisms that includebacteria, fungi, archaea, protists, plants (e.g., green algae), viruses,prions, parasites, and animals such as amoeba, plankton. Depending onthe context, microorganisms may be viewed as either harmful orbeneficial. In some cases, microorganisms may be harmful and lead toillness and disease in plants, animals or humans. Moreover, in additionto causing infections or diseases, undesired microbial growth may alsooccur in consumer products, such as food contamination. In other cases,microorganism growth is beneficial and is routinely exploited inbiotechnology, modern diagnostic technologies, in chemical processes(e.g., fermentation), in food and beverage preparation, in environmentaland industrial applications, and in maintaining and/or promoting humanhealth.

SUMMARY

Disclosed herein are methods of modulating microorganism activity withMSM. MSM is an organosulfur compound with the formula (CH₃)₂SO₂. Inparticular, disclosed herein is the surprising capability of MSM toenhance or inhibit microorganism activity, such as microorganism growthor survival, depending upon the concentration of MSM provided to themicroorganism (e.g., in the medium in which the organism is grown). MSMat a concentration of about 0.5% to about 5% by weight of medium or byweight of moisture content of the medium enhances microbial activitywhereas MSM at a concentration of about 6% to about 17% by weight ofmedium or by weight of moisture content of the medium inhibits microbialactivity.

Disclosed herein is the surprising discovery that MSM can both inhibitand enhance microbial activity, depending upon the concentration of MSM.For example, MSM concentrations between about 6 and about 17 percent byweight of medium (or of moisture content of medium inhibit microbialactivity by reducing or otherwise impacting the growth, survival rate(e.g., by causing or expediting cell deterioration or death, such asprogrammed cell death), metabolism, reproduction (e.g., gene expression,protein expression, signal transduction, transcription, translation,protein folding, etc.), proliferation, vitality, robustness, action,and/or function of the microorganism. In contrast, MSM concentrationsbetween about 0.04% to about 5% by weight enhance microbial activity,including enhancing microbial fermentation efficiency, microbial growthand/or culture efficiency.

As such, disclosed herein are methods of use of MSM to modulatemicrobial activity, such as to enhance or inhibit the activity ofmicroorganisms.

In some embodiments, a method of enhancing fermentation efficiency of amicroorganism is disclosed. For example, the method includes contactingmedium containing a microorganism capable of fermentation with MSM,wherein the MSM is provided at a concentration of about 0.5% to about 5%by weight of the medium or at a concentration of about 0.5% to about 5%by weight of the moisture content of the medium, wherein the MSMincreases the fermentation efficiency of the microorganism as comparedto the fermentation efficiency in the absence of MSM.

In some embodiments, in vitro methods for enhancing the growth of one ormore probiotic microorganisms are disclosed. In some examples, themethod comprises contacting one or more probiotic microorganisms with amedium capable of supporting growth of one or more probioticmicroorganisms; and providing MSM to the medium at about 0.4% to about5% by weight of the medium or by weight of a moisture content of themedium thereby enhancing growth of the one or more microorganisms invitro as compared to growth of the one or more microorganisms in vitroin the absence of MSM.

Also provided are methods for enhancing growth of a microorganism in adiagnostic test sample. In some examples, the method comprisescontacting the diagnostic test sample comprising one or moremicroorganisms with a medium capable of supporting growth of the one ormore microorganisms; providing MSM to the medium at a concentration ofabout 0.4% to about 5% by weight of the medium or by weight of amoisture content of the medium, thereby enhancing the growth of the oneor more microorganisms in the diagnostic test sample as compared togrowth of the one or more microorganisms in the absence of MSM.

Further disclosed are methods of inhibiting microbial activity. In someexamples, the method comprises selecting a medium that is susceptible toH1N1 influenza contamination; and contacting the medium with MSM at aconcentration of about 10% to about 16% of weight by volume, therebyinhibiting H1N1 influenza microbial activity.

The foregoing and other features of the disclosure will become moreapparent from the following detailed description of a severalembodiments.

DETAILED DESCRIPTION I. Overview of Several Embodiments

Disclosed herein is the surprising discovery that MSM can both inhibitand enhance microbial activity, depending upon the concentration of MSM.For example, MSM concentrations between about 6 and about 17 percent byweight of medium (or of moisture content of medium inhibit microbialactivity by reducing or otherwise impacting the growth, survival rate(e.g., by causing or expediting cell deterioration or death, such asprogrammed cell death), metabolism, reproduction (e.g., gene expression,protein expression, signal transduction, transcription, translation,protein folding, etc.), proliferation, vitality, robustness, action,and/or function of the microorganism. In contrast, MSM concentrationsbetween about 0.04% to about 5% by weight enhance microbial activity,including enhancing microbial fermentation efficiency, microbial growthand/or culture efficiency.

As such, disclosed herein are methods of use of MSM to modulatemicrobial activity, such as to enhance or inhibit the activity ofmicroorganisms.

In some embodiments, a method of enhancing fermentation efficiency of amicroorganism is disclosed. For example, the method includes contactingmedium containing a microorganism capable of fermentation with MSM,wherein the MSM is provided at a concentration of about 0.5% to about 5%by weight of the medium or at a concentration of about 0.5% to about 5%by weight of the moisture content of the medium, wherein the MSMincreases the fermentation efficiency of the microorganism as comparedto the fermentation efficiency in the absence of MSM.

In some examples, enhancing fermentation efficiency comprises an atleast 50% increase in alcohol, carbon dioxide or acid production in thepresence of MSM by the microorganism as compared to alcohol or acidproduction in the absence of MSM. For example, enhancing fermentationefficiency comprises an at least 50% increase in production of ethanol,methanol or a combination of thereof as compared to production ofethanol, methanol or a combination of thereof in the absence of MSM.

In some examples, enhancing fermentation efficiency comprises an atleast 50% increase in carbon dioxide production in the presence of MSMby the microorganism as compared to carbon dioxide production in theabsence of MSM, the microorganism is yeast and the method of enhancingfermentation is for the production of bread.

In some examples, enhancing fermentation efficiency comprises an atleast 50% increase in lactic acid production in the presence of MSM bythe microorganism as compared to lactic acid production in the absenceof MSM and the method of enhancing fermentation is for the production ofa dairy product.

In some embodiments, the method of enhancing fermentation efficiency isfor the production of beer, cider, wine, a biofuel, bread, dairy productor any combination thereof. In some examples, the microorganism is yeastand the method of enhancing fermentation is for the production of beer.In some examples, the microorganism is algae and the method of enhancingfermentation is for the production of biofuel.

In some embodiments, the concentration of MSM is about 0.5%. In someexamples, the medium comprises a sodium chloride concentration of lessthan 5% of total moisture content.

Also disclosed are in vitro methods for enhancing the growth of one ormore probiotic microorganisms. In some embodiments, the method comprisescontacting one or more probiotic microorganisms with a medium capable ofsupporting growth of one or more probiotic microorganisms; and providingMSM to the medium at about 0.4% to about 5% by weight of the medium orby weight of a moisture content of the medium thereby enhancing growthof the one or more microorganisms in vitro as compared to growth of theone or more microorganisms in vitro in the absence of MSM.

In some examples, the concentration of MSM is about 1% to about 3% ofthe weight of the medium or the moisture content of the medium.

In some examples, the one or more probiotic microorganisms comprisesLactobacillus acidophilus, Lactobacillus delbrueckii, Bacilluscoagulans, Lactobacillus rhamnosus, Bifidobacteruim bifidum or anycombination thereof.

In some examples, the medium comprises a probiotic-containing product,such as milk, yogurt, rice yogurt, frozen yogurt, chocolate, cheese,beer, wine, vinegar, sauerkraut or any combination thereof.

Also disclosed are methods for enhancing growth of a microorganism in adiagnostic test sample. In some examples, the method comprisescontacting the diagnostic test sample comprising one or moremicroorganisms with a medium capable of supporting growth of the one ormore microorganisms; providing MSM to the medium at a concentration ofabout 0.4% to about 5% by weight of the medium or by weight of amoisture content of the medium, thereby enhancing the growth of the oneor more microorganisms in the diagnostic test sample as compared togrowth of the one or more microorganisms in the absence of MSM.

Further disclosed are methods of inhibiting microbial activity. In someexamples, the method comprises selecting a medium that is susceptible toH1N1 influenza contamination; and contacting the medium with MSM at aconcentration of about 10% to about 16% of weight by volume, therebyinhibiting H1N1 influenza microbial activity. In some examples, themedium comprises a bodily fluid, a bodily tissue, or a surface. In someexamples, contacting the medium comprises spraying or wiping the mediumsusceptible to microbial contamination with MSM. In some examples, thesurface is a household surface, bedding, coverings, industrial equipmentor surface, blood, skin or a combination thereof. In some examples, MSMis provided in a composition, wherein said composition is free of bleachor free alcohol or consists essentially of water. In some examples, themethod further comprises sterilizing the medium after adding said MSM.In some examples, the medium is free from preservatives. In someexamples, the MSM inhibits the microbial activity by reducing growthrate of H1N1 influenza by at least 50% as compared to the growth rate ofH1N1 influenza in the absence of MSM.

II. Abbreviations and Terms

DMEM: Dulbecco's modified eagle mediumDMSO: Dimethyl sulfoxideDNA: Deoxyribonucleic acidELISA: Enzyme-linked immunosorbent assayIC₅₀: Inhibitory concentration 50LAB: Lactic acid bacteriaMIC: Minimum inhibitory concentration

MSM: Methylsulfonylmethane

PAGE: Polyacrylamide-gel electrophoresisPBS: Phosphate buffered salinePDA: Potato dextrose agarSDS: Sodium dodecyl sulfateTNTC: Too numerous to countTSB: Tryptic soy broth

The following explanations of terms and methods are provided to betterdescribe the present disclosure and to guide those of ordinary skill inthe art in the practice of the present disclosure. The singular forms“a,” “an,” and “the” refer to one or more than one, unless the contextclearly dictates otherwise. For example, the term “comprising abacterial cell” includes single or plural bacterial cells and isconsidered equivalent to the phrase “comprising at least one bacterialcell.” The term “or” refers to a single element of stated alternativeelements or a combination of two or more elements, unless the contextclearly indicates otherwise. As used herein, “comprises” means“includes.” Thus, “comprising A or B,” means “including A, B, or A andB,” without excluding additional elements.

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this disclosure belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. The materials, methods, and examples areillustrative only and not intended to be limiting. For example,conventional methods well known in the art to which a disclosedinvention pertains are described in various general and more specificreferences, including, for example, Sambrook et al., Molecular Cloning:A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989;Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., ColdSpring Harbor Press, 2001; Ausubel et al., Current Protocols inMolecular Biology, Greene Publishing Associates, 1992 (and Supplementsto 2000); Ausubel et al., Short Protocols in Molecular Biology: ACompendium of Methods from Current Protocols in Molecular Biology, 4thed., Wiley & Sons, 1999; Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, 1990; and Harlow and Lane,Using Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 1999; Loudon, Organic Chemistry, Fourth Edition, New York: OxfordUniversity Press, 2002, pp. 360-361, 1084-1085; Smith and March, March'sAdvanced Organic Chemistry: Reactions, Mechanisms, and Structure, FifthEdition, Wiley-Interscience, 2001; or Vogel, A Textbook of PracticalOrganic Chemistry, Including Qualitative Organic Analysis, FourthEdition, New York: Longman, 1978.

Additional terms commonly used in molecular genetics can be found inBenjamin Lewin, Genes V published by Oxford University Press, 1994 (ISBN0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of MolecularBiology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9);and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: aComprehensive Desk Reference, published by VCH Publishers, Inc., 1995(ISBN 1-56081-569-8).

Additional terms commonly used in chemistry can be found in Loudon,Organic Chemistry, Fourth Edition, New York: Oxford University Press,2002, pp. 360-361, 1084-1085; Smith and March, March's Advanced OrganicChemistry: Reactions, Mechanisms, and Structure, Fifth Edition,Wiley-Interscience, 2001; or Vogel, A Textbook of Practical OrganicChemistry, Including Qualitative Organic Analysis, Fourth Edition, NewYork: Longman, 1978.

Administration: To provide or give a subject a compound, such as MSM, byany effective route. Exemplary routes of administration include, but arenot limited to, injection (such as subcutaneous, intramuscular,intradermal, intraperitoneal, and intravenous), oral, sublingual,rectal, transdermal (such as topical), intranasal, vaginal andinhalation routes. A particular type of administration is topical.

Bacterial pathogen: A bacteria that causes disease (pathogenicbacteria). Examples of pathogenic bacteria for which MSM may be used tomodify include without limitation any one or more of (or any combinationof) Acinetobacter baumanii, Actinobacillus sp., Actinomycetes,Actinomyces sp. (such as Actinomyces israelii and Actinomycesnaeslundii), Aeromonas sp. (such as Aeromonas hydrophila, Aeromonasveronii biovar sobria (Aeromonas sobria), and Aeromonas caviae),Anaplasma phagocytophilum, Alcaligenes xylosoxidans, Acinetobacterbaumanii, Actinobacillus actinomycetemcomitans, Bacillus sp. (such asBacillus anthracis, Bacillus cereus, Bacillus subtilis, Bacillusthuringiensis, and Bacillus stearothermophilus), Bacteroides sp. (suchas Bacteroides fragilis), Bartonella sp. (such as Bartonellabacilliformis and Bartonella henselae, Bifidobacterium sp., Bordetellasp. (such as Bordetella pertussis, Bordetella parapertussis, andBordetella bronchiseptica), Borrelia sp. (such as Borrelia recurrentis,and Borrelia burgdorferi), Brucella sp. (such as Brucella abortus,Brucella canis, Brucella melintensis and Brucella suis), Burkholderiasp. (such as Burkholderia pseudomallei and Burkholderia cepacia),Campylobacter sp. (such as Campylobacter jejuni, Campylobacter coli,Campylobacter lari and Campylobacter fetus), Capnocytophaga sp.,Cardiobacterium hominis, Chlamydia trachomatis, Chlamydophilapneumoniae, Chlamydophila psittaci, Citrobacter sp. Coxiella burnetii,Corynebacterium sp. (such as, Corynebacterium diphtheriae,Corynebacterium jeikeum and Corynebacterium), Clostridium sp. (such asClostridium perfringens, Clostridium difficile, Clostridium botulinumand Clostridium tetani), Eikenella corrodens, Enterobacter sp. (such asEnterobacter aerogenes, Enterobacter agglomerans, Enterobacter cloacaeand Escherichia coli, including opportunistic Escherichia coli, such asenterotoxigenic E. coli, enteroinvasive E. coli, enteropathogenic E.coli, enterohemorrhagic E. coli, enteroaggregative E. coli anduropathogenic E. coli) Enterococcus sp. (such as Enterococcus faecalisand Enterococcus faecium) Ehrlichia sp. (such as Ehrlichia chafeensiaand Ehrlichia canis), Erysipelothrix rhusiopathiae, Eubacterium sp.,Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis,Gemella morbillorum, Haemophilus sp. (such as Haemophilus influenzae,Haemophilus ducreyi, Haemophilus aegyptius, Haemophilus parainfluenzae,Haemophilus haemolyticus and Haemophilus parahaemolyticus, Helicobactersp. (such as Helicobacter pylori, Helicobacter cinaedi and Helicobacterfennelliae), Kingella kingii, Klebsiella sp. (such as Klebsiellapneumoniae, Klebsiella granulomatis and Klebsiella oxytoca),Lactobacillus sp., Listeria monocytogenes, Leptospira interrogans,Legionella pneumophila, Leptospira interrogans, Peptostreptococcus sp.,Moraxella catarrhalis, Morganella sp., Mobiluncus sp., Micrococcus sp.,Mycobacterium sp. (such as Mycobacterium leprae, Mycobacteriumintracellulare, Mycobacterium avium, Mycobacterium bovis, andMycobacterium marinum), Mycoplasm sp. (such as Mycoplasma pneumoniae,Mycoplasma hominis, and Mycoplasma genitalium), Nocardia sp. (such asNocardia asteroides, Nocardia cyriacigeorgica and Nocardiabrasiliensis), Neisseria sp. (such as Neisseria gonorrhoeae andNeisseria meningitidis), Pasteurella multocida, Plesiomonasshigelloides. Prevotella sp., Porphyromonas sp., Prevotellamelaminogenica, Proteus sp. (such as Proteus vulgaris and Proteusmirabilis), Providencia sp. (such as Providencia alcalifaciens,Providencia rettgeri and Providencia stuartii), Pseudomonas aeruginosa,Propionibacterium acnes, Rhodococcus equi, Rickettsia sp. (such asRickettsia rickettsii, Rickettsia akari and Rickettsia prowazekii,Orientia tsutsugamushi (formerly: Rickettsia tsutsugamushi) andRickettsia typhi), Rhodococcus sp., Serratia marcescens,Stenotrophomonas maltophilia, Salmonella sp. (such as Salmonellaenterica, Salmonella typhi, Salmonella paratyphi, Salmonellaenteritidis, Salmonella cholerasuis and Salmonella typhimurium),Serratia sp. (such as Serratia marcesans and Serratia liquifaciens),Shigella sp. (such as Shigella dysenteriae, Shigella flexneri, Shigellaboydii and Shigella sonnei), Staphylococcus sp. (such as Staphylococcusaureus, Staphylococcus epidermidis, Staphylococcus hemolyticus,Staphylococcus saprophyticus), Streptococcus sp. (such as Streptococcuspneumoniae (for example chloramphenicol-resistant serotype 4Streptococcus pneumoniae, spectinomycin-resistant serotype 6BStreptococcus pneumoniae, streptomycin-resistant serotype 9VStreptococcus pneumoniae, erythromycin-resistant serotype 14Streptococcus pneumoniae, optochin-resistant serotype 14 Streptococcuspneumoniae, rifampicin-resistant serotype 18C Streptococcus pneumoniae,tetracycline-resistant serotype 19F Streptococcus pneumoniae,penicillin-resistant serotype 19F Streptococcus pneumoniae, andtrimethoprim-resistant serotype 23F Streptococcus pneumoniae,chloramphenicol-resistant serotype 4 Streptococcus pneumoniae,spectinomycin-resistant serotype 6B Streptococcus pneumoniae,streptomycin-resistant serotype 9V Streptococcus pneumoniae,optochin-resistant serotype 14 Streptococcus pneumoniae,rifampicin-resistant serotype 18C Streptococcus pneumoniae,penicillin-resistant serotype 19F Streptococcus pneumoniae, ortrimethoprim-resistant serotype 23F Streptococcus pneumoniae),Streptococcus agalactiae, Streptococcus mutans, Streptococcus pyogenes,Group A streptococci, Streptococcus pyogenes, Group B streptococci,Streptococcus agalactiae, Group C streptococci, Streptococcus anginosus,Streptococcus equismilis, Group D streptococci, Streptococcus bovis,Group F streptococci, and Streptococcus anginosus Group G streptococci),Spirillum minus, Streptobacillus moniliformi, Treponema sp. (such asTreponema carateum, Treponema petenue, Treponema pallidum and Treponemaendemicum, Tropheryma whippelii, Ureaplasma urealyticum, Veillonellasp., Vibrio sp. (such as Vibrio cholerae, Vibrio parahemolyticus, Vibriovulnificus, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrioalginolyticus, Vibrio mimicus, Vibrio hollisae, Vibrio fluvialis, Vibriometchnikovii, Vibrio damsela and Vibrio furnisii), Yersinia sp. (such asYersinia enterocolitica, and Yersinia pestis) and Xanthomonasmaltophilia among others.

In some embodiments, MSM is used to modify, such as increase or decreasethe biological activity of one or more of the organisms listed above.

Beta-lactam antibiotics: A class of antibiotic agents containing aβ-lactam nucleus in its molecular structure. Examples include thepenicillin, cephalosporin, monobactam, and carbapenem families ofantibiotics. Methicillin and Oxacillin are Beta-lactam antibiotics.

Biological activity: An expression describing the beneficial or adverseeffects of a substance on living matter. When the agent is a complexchemical mixture, this activity is exerted by the substance's activeingredient or pharmacophore, but can be modified by the otherconstituents. Activity is generally dosage-dependent and it is notuncommon to have effects ranging from beneficial to adverse for onesubstance when going from low to high doses. In one example, MSM alters,such as increase or decreases the biological activity of amicroorganism, such as bacteria.

Biofuel: A fuel derived from a metabolic product of a living organism.It is a renewable energy source, unlike other natural resources such aspetroleum, coal and nuclear fuels. A biodiesel fuel is adiesel-equivalent processed fuel derived from biological sources whichcan be used in unmodified diesel-engine vehicles. Biodiesels areattractive for fuels, and some other uses, because they have a low vaporpressure, are non-toxic, stable and do not deteriorate or detonate uponmild heating. Chemically, biodiesels are generally defined as the monoalkyl esters of long chain fatty acids derived from renewable lipidsources.

Bleach: A solution of approximately 3-6% sodium hypochlorite (NaClO),and oxygen bleach, which contains hydrogen peroxide or aperoxide-releasing compound such as sodium perborate, sodiumpercarbonate, sodium persulfate, tetrasodium pyrophosphate, or ureaperoxide together with catalysts and activators, e.g.,tetraacetylethylenediamine and/or sodium nonanoyloxybenzenesulfonate.Bleaching powder is calcium hypochlorite. Many bleaches have strongbactericidal properties, and are used for disinfecting and sterilizing.

Conditions that permit production: Any fermentation or culturingconditions that allow a microorganism to grow and/or produce a desiredproduct, such as alcohols and carbon dioxide or organic acids. Suchconditions usually include temperature ranges, levels of aeration, andmedia selection that, when combined, allow the microorganism to grow.Exemplary mediums include broths or gels. To determine if cultureconditions permit product production, the microorganism can be culturedfor 2, 4, 6, 8, 12, 24, 36, 48 or 72 hours and a sample can be obtainedand analyzed. For example, the cells in the sample or the medium inwhich the cells were grown can be tested for the presence of the desiredproduct. When testing for the presence of a product, assays can be used,such as those provided herein, including those presented in the Examplesbelow.

Contacting: Placement in direct physical association; including insolid, liquid and gas form. Contacting includes contact between onemolecule and another molecule. Contacting can occur in vitro withisolated cells, tissue or a solid surface (such as a household orindustrial surface) or in vivo by administering to a subject.

Control: Samples believed to be normal (e.g., representative activity orfunction in the absence of the variable being tested) as well aslaboratory values, even though possibly arbitrarily set, keeping in mindthat such values can vary from laboratory to laboratory. A control groupis practically identical to the treatment group, except for the singlevariable of interest whose effect is being tested, which is only appliedto the treatment group.

Culturing: Maintaining a cell in a medium that allows the organism tocontinue to live. For example, culturing includes incubating amicroorganism in a fermentation media, such as a fermentation broth orfermentation gel. One of ordinary skill in the art will appreciate thatthe time, temperature, and other physical conditions associated withculturing will depend upon the organism being cultured and the desiredoutcome from the culture. For example, a microorganism that is culturedto produce ethanol can be placed in a fermentation broth containing acarbohydrate source, various minerals and trace elements, as well as MSMand compounds useful for inducing the production, including less than 5%NaCl.

Decrease: To reduce the quality, amount, or strength of something. Inone example, administration of MSM decreases or reduces one or morebiological activities, such as growth, reproduction, proliferation,survival rate, metabolism, vitality, robustness, action, and/or functionof microorganisms by at least 10%, at least 20%, at least 50%, or evenat least 90%, including between 10% to 95%, 20% to 80%, 30% to 70%, 40%to 50%, such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%,98%, or 100%. For example, administration of MSM decreases or inhibitsbacterial growth, for example by at least 2-fold, for example at least3-fold or at least 4-fold, as compared to a control (such as bacterialgrowth in the absence of MSM or a reference value known to berepresentative of bacterial growth in a subject afflicted with abacterial infection). Such decreases can be measured using the methodsdisclosed herein as well as those known to one of ordinary skill in theart. In some embodiments, MSM is used to inhibit growth of specificmicroorganisms. In other embodiments, MSM is used to inhibit growth of awide range of microorganisms in certain media or products. In someembodiments, log-scale reductions are realized after the first 24 hours.

Dimethyl sulfoxide (DMSO): Dimethyl sulfoxide (DMSO), also known asmethylsulfinylmethane or methyl sulfoxide, is an organosulfur compoundwith the formula (CH₃)₂SO. This colorless liquid is a polar aproticsolvent that dissolves both polar and nonpolar compounds and is misciblein a wide range of organic solvents as well as water. It has adistinctive property of penetrating the skin very readily, so that onemay taste it soon after it comes into contact with the skin DMSO is wellknown as a nutritional supplement and as a pharmaceutical agent. One ofskill in the relevant art will be familiar with these uses. Variousgrades of DMSO are available commercially (for example, product No.472301 from Sigma-Aldrich, Corp., St. Louis, Mo.) and one of skill inthe art will be familiar with a source of DMSO.

Enhance or increase: To increase the quality, amount, or strength ofsomething. In one example, MSM increases or enhances the activity of amicroorganism, for example relative to activity in the absence of MSM.In a particular example, MSM increases the activity of a microorganism,such as enhancing the growth, reproduction, proliferation, survivalrate, metabolism, vitality, robustness, action, and/or function of amicroorganism by at least 10%, at least 20%, at least 50%, or even atleast 90%, including between 10% to 95%, 20% to 80%, 30% to 70%, 40% to50%, such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%,or 100%. The terms activity and growth are used interchangeably in somecontexts. In some examples, MSM is used to enhance growth of specificmicroorganisms. In other examples, MSM is used to enhance growth of awide range of microorganisms in certain media or products. In someexamples, enhancing microbial activity includes enhancing microbialproducts or microbial metabolites. For example, MSM increases orenhances fermentation efficiency or culturing efficiency such as by atleast 10%, at least 20%, at least 50%, or even at least 90%, includingbetween 10% to 95%, 20% to 80%, 30% to 70%, 40% to 50%, such as 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 100%. Suchincreases can be measured using the methods disclosed herein.

Fermentation: A process of deriving energy from the oxidation of organiccompounds, such as carbohydrates, and using an endogenous electronacceptor, which is usually an organic compound. During fermentation,pyruvate is metabolised to various different compounds. Homolacticfermentation is the production of lactic acid from pyruvate; alcoholicfermentation is the conversion of pyruvate into ethanol and carbondioxide; and heterolactic fermentation is the production of lactic acidas well as other acids and alcohols. Fermentation does not necessarilyhave to be carried out in an anaerobic environment. For example, even inthe presence of abundant oxygen, yeast cells prefer fermentation tooxidative phosphorylation, as long as sugars are readily available forconsumption. Sugars are a common substrate of fermentation, and typicalexamples of fermentation products are ethanol, lactic acid, andhydrogen. However, more exotic compounds can be produced byfermentation, such as butyric acid and acetone. Yeast carries outfermentation in the production of ethanol in beers, wines and otheralcoholic drinks, along with the production of large quantities ofcarbon dioxide.

Fermentation Broth: Any medium that supports microorganism life (forinstance, a microorganism that is actively metabolizing carbon). Afermentation medium usually contains a carbon source. The carbon sourcecan be anything that can be utilized, with or without additionalenzymes, by the microorganism for energy.

Fermentation efficiency: An expression of how much fermentation product,such as alcohol, lactic acid, micro-organisms or other desiredfermentation product, was produced relatively to a control (such as inthe absence of MSM) or to an amount that could be theoreticallyproduced.

Fermentation media: Any substance used to culture cells, such asmammalian cells and microorganisms. Fermentation media includes anygrowth medium (e.g., broth or gel) which supports microorganism life(e.g., a microorganism that is actively metabolizing carbon). Afermentation medium usually contains a carbon source, such as glucose,xylose, cellulosic material and the like. The carbon source can beanything that can be utilized, with or without additional enzymes, bythe microorganism for energy.

Fungal pathogen: A fungus that causes disease. Examples of fungalpathogens for which MSM can be used to modify include without limitationany one or more of (or any combination of) Trichophyton rubrum, T.mentagrophytes, Epidermophyton floccosum, Microsporum canis,Pityrosporum orbiculare (Malassezia furfur), Candida sp. (such asCandida albicans), Aspergillus sp. (such as Aspergillus fumigatus,Aspergillus flavus, Aspergillus glaucus, Aspergillus nidulans,Aspergillus oryzae, Aspergillus terreus, Aspergillus ustus, Aspergillusversicolor and Aspergillus clavatus), Cryptococcus sp. (such asCryptococcus neoformans, Cryptococcus gattii, Cryptococcus laurentii andCryptococcus albidus), Coccidioides sp., Histoplasma sp. (such asHistoplasma capsulatum), Pneumocystis sp. (such as Pneumocystisjirovecii), Stachybotrys sp. (such as Stachybotrys chartarum),Paracoccidioides, Blastomyce, Fusarium, Sporothrix, Trichosporon,Rhizopus, Pseudallescheria, Paecilomyces, Alternaria, Curvularia,Exophiala, Wangiella, Penicillium, and Cephalosphorium. In someembodiments, MSM is administered to inhibit or prevent an infection ordisorder associated with one or more of the aforementioned fungalpathogens.

Incubating: A term that includes a sufficient amount of time for anagent, such as MSM, to interact with a cell or tissue.

Inhalant device: A device capable of delivering a composition to asubject, for example to a subject's lung tissue. For example, aninhalant device may be an inhaler, a nebulizer or a ventilator. Inhalantdevices described herein are constructed from a material adapted forcontacting DMSO and/or MSM. In some embodiments, an inhalant device isdisposable or replaceable. Inhalant devices described herein areconfigured to deliver a DMSO or MSM containing composition to directlycontact bacterial pathogens in a subject's lung tissue. Inhalant devicesare configured to generate particles of a composition that range insize. In some embodiments, an inhalant device is configured to generateparticles of a composition that range in size from about 0.1 μm to about10 μm or from about 0.5 μm to about 5 μm.

Inhibiting microbial activity or Inhibiting an Infection or Disease: Thephrase “inhibiting microbial activity” refers to reducing the growth,reproduction, proliferation, survival rate, metabolism, vitality,robustness, action, and/or function of microorganisms. The phrase“inhibiting or treating an infection, disease or a condition” refers topreventing or reducing the full development of an infection, disease orcondition, for example, in a subject who is at risk for developing aninfection, such as a bacterial infection. “Treatment” refers to atherapeutic intervention that ameliorates a sign or symptom of a diseaseor pathological condition after it has begun to develop. As used herein,the term “ameliorating,” with reference to a disease, pathologicalcondition or symptom, refers to any observable beneficial effect of thetreatment. The beneficial effect can be evidenced, for example, by adelayed onset of clinical symptoms of the infection/disease in asusceptible subject, a reduction in severity of some or all clinicalsymptoms of the infection/disease, a slower progression of theinfection/disease, a reduction in the number of relapses of theinfection/disease, an improvement in the overall health or well-being ofthe subject, or by other parameters well known in the art that arespecific to the particular infection/disease, such as a particularbacterial infection.

Medium or media: An environment containing or suitable for supportingmicroorganisms, including, but not limited to, broths, agar, cultures,foods, beverages, cell suspensions, biological tissue, biologicalfluids, inorganic surfaces, organic surfaces, substrates, living cells,host cells, diagnostic assays, and other solid, liquid, matrix,gelatinous, or gaseous environments.

Methylsulfonylmethane (MSM): An organosulfur compound with the formula(CH₃)₂SO₂. MSM has largely been marketed and sold as a dietarysupplement. MSM is also known as DMSO₂, Dimethyl sulfone and methylsulfone.

MSM is structurally related to dimethyl sulfoxide (DMSO), but thebehavior of these two is different. DMSO is a highly polar solvent andan excellent ligand, with water-like dissolving properties whereas MSMis less polar and less reactive. MSM is also a metabolite of DMSO. MSMhas the following chemical structure:

Microorganism: A member of the prokaryotic or eukaryotic microbialspecies from the domains Archaea, Bacteria, and Eucarya, the latterincluding yeast and filamentous fungi, protozoa, algae, or higherProtista. The terms “microbial cells” and “microbes” are usedinterchangeably with the term microorganism. Microbes can includewild-type, genetically-engineered or modified organisms. Microorganismsinclude viruses, prions, parasites, fungi, mold, yeast and bacteria. Insome embodiments, MSM is used to enhance the activity of a wide spectrumof microorganisms including, but not limited to, viruses, prions,parasites, fungi, mold, yeast, algae and bacteria. In other embodiments,MSM is used to inhibit the activity of microorganisms, including, butnot limited to, fungi, mold, yeast, bacteria and viruses.

Modulate or modulating: To adjust, alter, regulate an activity, a degreeor rate of such including an increase or a decrease in biologicalactivity of a molecule. In one example, MSM is administered to modulate,either increase or decrease microbial activity, such as bacterialgrowth.

Parasite: An organism that lives inside humans or other organisms actingas hosts (for the parasite). Parasites are dependent on their hosts forat least part of their life cycle. Parasites are harmful to humansbecause they consume needed food, eat away body tissues and cells, andeliminate toxic waste, which makes people sick. Examples of fungalpathogens for use in accordance with the disclosed methods andcompositions include without limitation any one or more of (or anycombination of) Malaria (Plasmodium falciparum, P. vivax, P. malariae),Schistosomes, Trypanosomes, Leishmania, Filarial nematodes,Trichomoniasis, Sarcosporidiasis, Taenia (T. saginata, T. solium),Leishmania, Toxoplasma gondii, Trichinelosis (Trichinella spiralis) orCoccidiosis (Eimeria species). MSM may be used to inhibit or preventactivity of one or more of the organisms listed above.

Pharmaceutical composition: A chemical compound or composition capableof inducing a desired therapeutic or prophylactic effect when properlyadministered to a subject. A pharmaceutical composition can include atherapeutic agent, a diagnostic agent or a pharmaceutical agent. Atherapeutic or pharmaceutical agent is one that alone or together withan additional compound induces the desired response (such as inducing atherapeutic or prophylactic effect when administered to a subject). In aparticular example, a pharmaceutical agent is an agent thatsignificantly reduces one or more symptoms associated with an infection,such as a bacterial or viral infection. In some embodiments, atherapeutic agent is an antibiotic agent, such as methicillin oroxacillin.

Pharmaceutically Acceptable Carriers or vehicles: The pharmaceuticallyacceptable carriers (vehicles) useful in this disclosure areconventional. Remington's Pharmaceutical Sciences, by E. W. Martin, MackPublishing Co., Easton, Pa., 19th Edition (1995), describes compositionsand compositions suitable for pharmaceutical delivery of one or moretherapeutic compounds or molecules, such as one or more peptidesprovided herein. In general, the nature of the carrier will depend onthe particular mode of administration being employed. For instance,parenteral compositions usually comprise injectable fluids that includepharmaceutically and physiologically acceptable fluids such as water,physiological saline, balanced salt solutions, aqueous dextrose,glycerol or the like as a vehicle. In a particular embodiment thecarrier is one that allows the therapeutic compound to cross theblood-brain barrier. For solid compositions (for example, powder, pill,tablet, or capsule forms), conventional non-toxic solid carriers caninclude, for example, pharmaceutical grades of mannitol, lactose,starch, or magnesium stearate. In addition to biologically-neutralcarriers, pharmaceutical compositions to be administered can containminor amounts of non-toxic auxiliary substances, such as wetting oremulsifying agents, preservatives, and pH buffering agents and the like,for example sodium acetate or sorbitan monolaurate.

Prebiotic: A non-digestible food ingredient that stimulates growthand/or activity of bacteria in the digestive tract which are beneficialto the health of the body. Typically, prebiotics are carbohydrates (suchas oligosaccharides); however, non-carbohydrates are also sources ofsuch ingredients. Prebiotics can be short-chain, long-chain, and/orfull-spectrum prebiotics. Short-chain prebiotics (such asoligofructose), contain 2-8 links per saccharide molecule, are typicallyfermented more quickly in the right-side of the colon providingnourishment to the bacteria in that area. Longer-chain prebiotics (suchas inulin) contain 9-64 links per saccharide molecule, and tend to befermented more slowly, nourishing bacteria predominantly in theleft-side colon. Full-spectrum prebiotics provide the full range ofmolecular link-lengths from 2-64 links per molecule, and nourishbacteria throughout the colon (such as oligofructose-enriched inulin,OEI). In some examples, a prebiotic increases the number and/or activityof bifidobacteria and lactic acid bacteria. Bifidobacteria and thelactic acid bacteria (lactobacillus or LABs) are bacteria which improvedigestion (including enhancing mineral absorption) and the effectivenessand intrinsic strength of the immune system. A product that stimulatesbifidobacteria, such as MSM, is considered a bifidogenic factor.Traditional dietary sources of prebiotics include soybeans, inulinsources (such as Jerusalem artichoke, jicama, and chicory root), rawoats, unrefined wheat, unrefined barley, garlic, leeks, onion,asparagus, banana and yacon. Prebiotic oligosaccharides are increasinglyadded to foods for their health benefits. Some oligosaccharides that areused in this manner are fructooligosaccharides (FOS),xylooligosaccharides (XOS), polydextrose and galactooligosaccharides(GOS). Some monosaccharides such as tagatose are also used sometimes asprebiotics. As used herein, MSM is a prebiotic.

Probiotic: A microorganism that confers a health benefit on the host,including, but not limited to, conferring protection from or treatmentof illness or undesired effects. Probiotics can confer health benefitsto a product, such as increasing the nutritional quality of edibleproducts, Probiotics include beneficial bacteria, such as lactic acidbacteria (such as Lactobacillus bulgaricus, Lactobacillus rhamnosus,Lactobacillus casei and Lactobacillus johnsonii) and bifidobacteria(such as Lactobacillus bifidus) which are the most common types ofmicrobes used as probiotics; but certain yeasts and bacilli may also beprobiotics. Probiotics are commonly consumed as part of fermented foods;such as in yogurt, soy products, or as dietary supplements. Liveprobiotic cultures are available in fermented dairy products andprobiotic fortified foods. However, tablets, capsules, powders andsachets containing the bacteria in freeze dried form are also available.Exemplary probiotic strains include, but are not limited to Bacilluscoagulans GBI-30, 6086 (Ganeden Biotech), Bifidobacterium LAFTI® B94(Institut-Rosell-Lallemand), Lactobacillus acidophilus LAFTI® L10(Institut-Rosell-Lallemand), Lactobacillus casei LAFTI® L26(Institut-Rosell-Lallemand), Bifidobacterium animalis subsp. lactisBB-12, Bifidobacterium breve (Yakult), Bifidobacterium infantis 35624(Procter & Gamble), Bifidobacterium animalis subsp. lactis HN019(Danisco), Bifidobacterium longum BB536 (Morinaga Milk Industry),Lactobacillus acidophilus DDS-1 (Nebraska Cultures), Lactobacillusacidophilus LA-5, Lactobacillus acidophilus NCFM (Danisco),Lactobacillus casei DN114-001 (Danone), Lactobacillus casei 431,Lactobacillus casei F19 (Arla Foods), Lactobacillus casei (Yakult),Lactobacillus paracasei St11 (or NCC2461, Nestlé), Lactobacillusjohnsonii La1 (Lactobacillus LC1, Lactobacillus johnsonii NCC533,Nestlé), Lactococcus lactis L1A (Norrmejerier), Lactobacillus plantarum299v (Probi), Lactobacillus reuteri ATTC 55730 (Lactobacillus reuteriSD2112, BioGaia Biologics), Lactobacillus rhamnosus ATCC 53013 (Valio),Lactobacillus rhamnosus LB21 (Norrmejerier), Bifidobacterium bifidum,Lactobacillus gasseri PA16/8, Bifidobacterium bifidum MF20/5,Bifidobacterium longum SP07/3, Streptococcus thermophilus, Lactobacillussalivarius, Bifidobacterium longum Rosell-175, Lactococcus lactisRosell-1058, Bifidobacterium breve Rosell-70, Lactobacillus rhamnosusRosell-11, Lactobacillus acidophilus Rosell-52, Bifidobacterium bifidumrosell-71, Bacillus subtilis var natto, Lactobacillus paracasei,Enterococcus faecium, Bifidobacterium animalis, Lactobacillusdelbrueckii, and Saccharomyces cerevisiae.

Quantitating: Determining or measuring a quantity (such as a relativequantity) of a molecule or the activity of a molecule, such as thequantity of analyte present in a sample.

Stem cell: A cell that has the ability to self replicate indefinitelyand that, under the right conditions, or given the right signals, candifferentiate into some or all of the different cell types that make upan organism. Stem cells have the potential to develop into mature,differentiated cells, such as heart cells, skin cells, or nerve cells.The fertilized egg is a stem cell because it has the potential togenerate all the cells and tissues that make up an embryo and thatsupport its development in utero. Adult mammals include more than 200kinds of cells, for instance, neurons, myocytes, epithelial cells,erythrocytes, monocytes, lymphocytes, osteocytes, and chondrocytes.Other cells that are essential for embryonic development but are notincorporated into the body of the embryo include the extraembryonictissues, placenta, and umbilical cord. All of these cells are generatedfrom a single fertilized egg.

Pluripotent cells can give rise to cells derived from all threeembryonic germ layers—mesoderm, endoderm, and ectoderm. Thus,pluripotent cells have the potential to give rise to any type of cell.Unipotent stem cells are capable of differentiating along only onelineage. Embryonic stem cells are pluripotent cells derived from theblastocyst. Adult stem cells are undifferentiated cells found in adifferentiated tissue that can replicate and become specialized to yieldall of the specialized cell types of the tissue from which itoriginated. Adult stem cells are capable of self-renewal for thelifetime of the organism. Sources of adult stem cells have been found inthe bone marrow, blood stream, cornea, retina, dental pulp, liver, skin,gastrointestinal tract, and pancreas. MSM is used herein to increase theculture efficiency, stability and/or viability of stem cells.

Sterilization: A process that eliminates (removes) or kills all forms oflife, including transmissible agents (such as fungi, bacteria, viruses,spore forms, etc.) present on a surface, contained in a fluid, inmedication, or in a compound such as biological culture media.Sterilization can be achieved methods known to one of ordinary skill inthe art, including by applying the proper combinations of heat,chemicals, irradiation, high pressure, and filtration.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes human and non-human mammals.

Symptom and sign: Any subjective evidence of disease or of a subject'scondition, e.g., such evidence as perceived by the subject; a noticeablechange in a subject's condition indicative of some bodily or mentalstate. A “sign” is any abnormality indicative of disease, discoverableon examination or assessment of a subject. A sign is generally anobjective indication of disease. Signs include, but are not limited toany measurable parameters such as tests for detecting a disorder ordisease, such as a bacterial or viral infection. In one example,reducing or inhibiting one or more symptoms or signs associated with abacterial or viral infection, includes reducing or inhibiting bacterialgrowth or viral infection by a desired amount, for example by at least20%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, at least 98%, or even at least 100%, as compared tothe bacterial growth or viral infectivity in the absence of MSM.

Therapeutically effective amount or concentration: An amount of acomposition that alone, or together with an additional therapeuticagent(s) sufficient to achieve a desired effect in a subject, or in acell, being treated with the agent. The effective amount of the agentwill be dependent on several factors, including, but not limited to thesubject or cells being treated, and the manner of administration of thetherapeutic composition. In one example, a therapeutically effectiveamount or concentration is one that is sufficient to preventadvancement, delay progression, or to cause regression of a disease, orwhich is capable of reducing symptoms caused by a condition or disease.

In one example, a desired effect is to reduce or inhibit one or moresymptoms associated with the disease. The one or more symptoms do nothave to be completely eliminated for the composition to be effective.For example, a composition can decrease the sign or symptom by a desiredamount, for example by at least 20%, at least 50%, at least 80%, atleast 90%, at least 95%, at least 98%, or even at least 100%, ascompared to the sign or symptom in the absence of MSM. In one particularexample, a desired response is to reduce or inhibit microorganismactivity (such as bacterial growth) by a desired amount, for example byat least 20%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or even at least 100%, ascompared to microorganism activity in the absence of MSM.

A therapeutically effective amount of a disclosed pharmaceuticalcomposition can be administered in a single dose, or in several doses,for example daily, during a course of treatment. However, thetherapeutically effective amount can depend on the subject beingtreated, the severity and type of the condition being treated, and themanner of administration. A therapeutically effective amount of an agentcan be measured as the concentration (moles per liter or molar-M) of theagent in blood (in vivo) or a buffer (in vitro) that produces thedesired effect(s). Alternatively, a therapeutically effective amount ofan agent can be measured as the amount administered to a subject perbody weight of the subject, for example, mg agent/kg body weight.

Untreated cell: A cell that has not been contacted with a desired agent,such as MSM. In an example, an untreated cell is a cell that receivesthe vehicle in which MSM was delivered.

Virus: A microscopic infectious organism that reproduces inside livingcells. A virus consists essentially of a core of nucleic acid surroundedby a protein coat, and has the ability to replicate only inside a livingcell. “Viral replication” is the production of additional virus by theoccurrence of at least one viral life cycle. A virus may subvert thehost cells' normal functions, causing the cell to behave in a mannerdetermined by the virus. For example, a viral infection may result in acell producing a cytokine, or responding to a cytokine, when theuninfected cell does not normally do so. In some examples, a virus is apathogen.

Specific examples of viral pathogens that might be treated in accordancewith the disclosed methods and compositions include without limitationany one or more of (or any combination of); Arenaviruses (such asGuanarito virus, Lassa virus, Junin virus, Machupo virus and Sabia),Arteriviruses, Roniviruses, Astroviruses, Bunyaviruses (such asCrimean-Congo hemorrhagic fever virus and Hantavirus), Barnaviruses,Birnaviruses, Bornaviruses (such as Borna disease virus), Bromoviruses,Caliciviruses, Chrysoviruses, Coronaviruses (such as Coronavirus andSARS), Cystoviruses, Closteroviruses, Comoviruses, Dicistroviruses,Flaviruses (such as Yellow fever virus, West Nile virus, Hepatitis Cvirus, and Dengue fever virus), Filoviruses (such as Ebola virus andMarburg virus), Flexiviruses, Hepeviruses (such as Hepatitis E virus),human adenoviruses (such as human adenovirus A-F), human astroviruses,human BK polyomaviruses, human bocaviruses, human coronavirus (such as ahuman coronavirus HKU1, NL63, and OC43), human enteroviruses (such ashuman enterovirus A-D), human erythrovirus V9, human foamy viruses,human herpesviruses (such as human herpesvirus 1 (herpes simplex virustype 1), human herpesvirus 2 (herpes simplex virus type 2), humanherpesvirus 3 (Varicella zoster virus), human herpesvirus 4 type 1(Epstein-Barr virus type 1), human herpesvirus 4 type 2 (Epstein-Barrvirus type 2), human herpesvirus 5 strain AD169, human herpesvirus 5strain Merlin Strain, human herpesvirus 6A, human herpesvirus 6B, humanherpesvirus 7, human herpesvirus 8 type M, human herpesvirus 8 type Pand Human Cyotmegalovirus), human immunodeficiency viruses (HIV) (suchas HIV 1 and HIV 2), human metapneumoviruses, human papillomaviruses(such as human papillomavirus-1, human papillomavirus-18, humanpapillomavirus-2, human papillomavirus-54, human papillomavirus-61,human papillomavirus-cand90, human papillomavirus RTRX7, humanpapillomavirus type 10, human papillomavirus type 101, humanpapillomavirus type 103, human papillomavirus type 107, humanpapillomavirus type 16, human papillomavirus type 24, humanpapillomavirus type 26, human papillomavirus type 32, humanpapillomavirus type 34, human papillomavirus type 4, humanpapillomavirus type 41, human papillomavirus type 48, humanpapillomavirus type 49, human papillomavirus type 5, humanpapillomavirus type 50, human papillomavirus type 53, humanpapillomavirus type 60, human papillomavirus type 63, humanpapillomavirus type 6b, human papillomavirus type 7, humanpapillomavirus type 71, human papillomavirus type 9, humanpapillomavirus type 92, and human papillomavirus type 96), humanparainfluenza viruses (such as human parainfluenza virus 1-3), humanparechoviruses, human parvoviruses (such as human parvovirus 4 and humanparvovirus B19), human respiratory syncytial viruses, human rhinoviruses(such as human rhinovirus A and human rhinovirus B), humanspumaretroviruses, human T-lymphotropic viruses (such as humanT-lymphotropic virus 1 and human T-lymphotropic virus 2), Human polyomaviruses, Hypoviruses, Leviviruses, Luteoviruses, Lymphocyticchoriomeningitis viruses (LCM), Marnaviruses, Narnaviruses, Nidovirales,Nodaviruses, Orthomyxoviruses (such as Influenza viruses),Partitiviruses, Paramyxoviruses (such as Measles virus and Mumps virus),Picornaviruses (such as Poliovirus, the common cold virus, and HepatitisA virus), Potyviruses, Poxviruses (such as Variola and Cowpox),Sequiviruses, Reoviruses (such as Rotavirus), Rhabdoviruses (such asRabies virus), Rhabdoviruses (such as Vesicular stomatitis virus,Tetraviruses, Togaviruses (such as Rubella virus and Ross River virus),Tombusviruses, Totiviruses, Tymoviruses, and Noroviruses among others.

In some embodiments, MSM is used to inhibit a biological activity of oneor more of the viruses listed above.

Yeast: A eukaryotic microorganism classified in the Kingdom Fungi, withabout 1,500 species described. Most reproduce asexually by budding,although a few reproduce by binary fission. Yeasts generally areunicellular, although some species may become multicellular through theformation of a string of connected budding cells known as pseudohyphae,or false hyphae. Exemplary yeasts that can be used in the disclosedmethods and compositions include but are not limited to Saccharomycescerevisiae, Candida albicans, Schizosaccharomyces pombe, Pichia,Cryptococcus, Zygosaccharomyces, Torulopsis, Hansenula, andDebaryomyces.

III. Compositions of MSM

Disclosed herein are compositions of MSM for use in modulating microbialactivity, such as enhancing or decreasing microbial activity. In someembodiments, a composition of MSM for use in enhancing microbialactivity includes about 0.02% to about 5% MSM by weight of the medium(such as culture medium) or by weight of the moisture content of themedium (such as culture medium), such as about 0.04% to about 4%, about1% to about 3%, including about 0.02%, about 0.03%, about 0.04%, about0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%,about 3%, about 0.5%, about 1%, about 2%, about 2.5%, about 3%, about 4%or about 5% of the weight of the medium or the moisture content of themedium. In some examples, the percentages of MSM provided herein arecalculated from the amount of a polar solvent, for example water in aproduct. By way of example, a composition with 5% MSM by weight of themedium would contain 5 grams of MSM per 100 grams or medium or acomposition with 5% MSM by weight of the moisture content of the mediumwould contain 5 grams of MSM per 100 grams or the polar solvent in themedium, excluding solids.

In some embodiments, the disclosed compositions include a medium capableof supporting growth of a microorganism, a microorganism, and MSM. Insome examples, a medium includes one or more of the following:probiotic-containing products, dairy products, milk, yogurt, riceyogurt, frozen yogurt, chocolate, cheese, fermented beverages (such asbeer, cider, wine) and water. In some examples, the medium also includesother products, edible or not, that benefit from enhanced microbialactivity.

In some embodiments, enhancing microbial activity include enhancing thefermentation of a microorganism. Thus in some particular examples, acomposition includes a medium capable of supporting growth offermentative microorganisms, a fermentative microorganism and MSM. Insome examples, MSM is provided at a concentration of about 0.04% toabout 5%, such as about 0.1% to about 4%, 0.5% to about 3%, about 1% toabout 2%, including about 0.04%, to about 0.05%, about 0.06%, about0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.3%, about 0.5%,about 0.7%, about 1%, about 1.5%, about 2.0%, about 2.5%, about 3.0%,about 4%, or about 4.5% MSM by weight of medium or by weight of themoisture content of the medium, wherein the concentration of MSM iseffective for enhancing the fermentation of the microorganisms. In someembodiments, the disclosed compositions are used to produce a fermentedbeverage, such as beer, cider and/or wine. In some embodiments, acomposition for enhancing fermentation efficiency includes MSM added toyeast packages to generate rapid or quick-activating yeast for home orcommercial use.

In some embodiments, enhancing microbial activity include enhancing thegrowth of probiotics. Thus, in some examples, a composition forenhancing the growth of probiotics includes a medium capable ofsupporting growth of probiotics and MSM at a concentration of about0.04% to about 5% by weight of the medium or by weight of the moisturecontent of the medium, wherein the concentration of MSM is effective forenhancing the activity (e.g., growth) of the probiotics. Further, acomposition for enhancing the growth of the probiotics includes about0.04% to about 5% MSM, such as about 0.1% to about 4%, 0.5% to about 3%,about 1% to about 2%, including about 0.04%, to about 0.05%, about0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.3%,about 0.5%, about 0.7%, about 1%, about 1.5%, about 2.0%, about 2.5%,about 3.0%, about 4%, or about 4.5% MSM by weight of medium or by weightof moisture content of the medium.

In some embodiments, enhancing microbial activity includes enhancingmicrobial production of biofuel. Thus, in some examples, a compositionfor enhancing microbial production of biofuel includes a medium capableof supporting growth of algae, algae capable of producing a biofuel andMSM at a concentration of about 0.04% to about 5%, such as about 0.1% toabout 4%, 0.5% to about 3%, about 1% to about 2%, including about 0.04%,to about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%,about 0.1%, about 0.3%, about 0.5%, about 0.7%, about 1%, about 1.5%,about 2.0%, about 2.5%, about 3.0%, about 4%, or about 4.5% MSM byweight of medium or by weight of the moisture content of the medium,wherein the concentration of MSM is effective for enhancing theproduction of the biofuel from the algae. In other examples, acomposition includes algae and MSM, and optionally other ingredients forenhancing algae growth. In several embodiments, the composition isuseful to enhance algae activity for biofuel, algal farming,aquaculture, medicaments, etc. In one embodiment, the method comprisesexposing algae to MSM at e.g., a concentration of about 0.04% to about5% by weight of medium or by weight of the moisture content of themedium.

Compositions of MSM for inhibiting microbial activity are disclosed. Insome embodiments, a composition of MSM for inhibiting microbial activityincludes about 6% to about 17%, such as about 7% to about 15%, about 10%to about 12%, such as about 6%, about 7%, about 8%, about 9%, about 10%,about 11%, about 13%, about 14%, about 15%, or about 16% MSM by weightof medium or by weight of the moisture content of the medium, whereinthe concentration of MSM is effective for inhibiting microbial activity,including, but not limited to microbial growth, infection rate, or acombination thereof.

It is contemplated that any of the disclosed compositions including MSMto modulate microbial activity have a sodium chloride concentration ofless than 5% of total moisture content of the medium, such as about 1%to about 3% sodium chloride, including 0%, 0.1%, 0.3%, 0.5%, 0.75%, 1%,2%, 2.5%, 3% or 4%. In some examples, a disclosed composition of MSM ispreservative free. For example, MSM is added to a food, cosmetic orbeverage product that requires or desires an all-natural ingredientlist. In some embodiments, a composition of MSM consists or consistsessentially of MSM and all-natural non-toxic ingredients. In otherexamples, a disclosed composition of MSM includes one or more additionalpreservatives. Preservatives include, but are not limited to one or acombination of the following: formaldehyde, potassium sorbate,methylparaben, methylchloroisothiazolinone, phthalates, cocamidopropylbetain, parabens, decyl polyglucose, polyaminopropyl biguanide,phenoxyethanol, sodium laureth sulfate, tetrasodium EDTA, decylglucoside, polyethylene glycol, and propylene glycol.

In several embodiments, MSM is used to extend the shelf-life of productsand is capable of reducing microbial activity by at least 2-, 3-, 4-,5-, 10-, 25-, 50-, 100-, 1000-fold as compared to products with no MSMor as compared to products with less effective antimicrobial agents. Inother embodiments, MSM is able to achieve comparable levels ofantimicrobial activity as compared to agents that produce undesired sideeffects. Thus, in one embodiment, MSM can be used instead of anundesired preservative. In several embodiments, compositions includepreservative-free or reduced preservative formulations comprising MSM.

In several embodiments, MSM is used in products (for example, cosmetics)that have an acidic, basic or neutral pH. Because MSM can inhibitmicrobial activity, cosmetics and other products can have moreflexibility in pH selection. Thus, a pH that is optimal for the productcan be selected. In several embodiments, products comprising MSM do notrequire refrigeration and may be stored at room temperature. In otherembodiments, products comprising MSM do not require sterilization,including but not limited to sterilization via chemicals, heating,radiation, filtration or ultraviolet light.

In several embodiments, disclosed compositions include MSM in additionto one or more thickening agents, emollients, and/or aromatic agents. Insome embodiments, a product or other medium is supplemented withcontinuous or periodic additions of MSM to, for example, extend theinhibitory or stimulatory actions of MSM.

In certain embodiments, the addition of MSM is an effectiveantimicrobial agent. For example, in one embodiment, a compositioncomprising MSM has the same or enhanced antimicrobial effect as comparedto a formulation with no MSM. In other embodiments, MSM serves as anantibacterial agent. In certain embodiments, MSM is used as a substitutefor a chemical food preservative. In still other embodiments, MSM may beused in combination with a preservative. In certain such embodiments,the use of MSM reduces the amount of, or altogether replaces,traditional preservatives. In some embodiments, MSM may increase theshelf life of a product, including products that traditionally would nothave a preservative. In yet other embodiments, MSM serves as a virucide,fungicide and/or bacterioside. In further embodiments, MSM isbacteriostatic. In some embodiments, MSM is a broad spectrum inhibitorof microbial activity. In other embodiments, MSM selectively kills acertain kingdom, genus or species. In some embodiments, MSM selectivelyinhibits aerobic bacteria. In other embodiments, MSM selectivelyinhibits anaerobic bacteria. In some embodiments, MSM selectivelyinhibits gram-positive bacteria. In other embodiments, MSM selectivelyinhibits gram-negative bacteria.

In several embodiments, MSM is used to inhibit the growth ofmicroorganisms, including those found in cosmetics, health and beautyaids, parenterals, topically used products, and oral products. Inseveral preferred embodiments, MSM is used to inhibit the growth ofmicroorganisms in products packaged in single or multiple-dosecontainers. MSM formulations according to several of the embodimentsdescribed herein are in any suitable form, including but not limited to,powder, cream, liquid, paste, solid or gel form.

In several embodiments, MSM is added to cosmetic products susceptible tomicrobial contamination. Cosmetics may include, but are not limited to,lipstick, lip gloss, lip liner, lip plumper, lip balm, lip conditionerand lip boosters, foundation, powder, rouge, blush, bronzer, mascara,eye liner, eye shadow, eye shimmer, glitter eye pencils, eyebrow pencil,nail polish, concealer, skin care products, creams, lotions, serums,moisturizer, sunscreen, skin repair products (e.g., for acne, sunburn,wrinkles, dark circles), and sunscreen.

In several embodiments, MSM is added to a cosmetic cream matrixsusceptible to microbial contamination. In some such embodiments, thecream includes jojoba, aloe vera, cocoa butter, shea butter, coconutoil, or combinations thereof.

In several embodiments, MSM is added to personal care productssusceptible to microbial contamination. Such products include productsused for daily moisturizing needs, products to treat psoriasis oreczema, products to treat dry or itchy skin, products to treat sun andwind burn, pre- and post-shave products, massage oils or creams,personal lubricants, acne treatment products and exfoliants oremollients. According to other embodiments, MSM is added to products tosoften skin on the hands or feet (such as calluses), after swim skincare products, make-up remover, children's skin lotion, and diaper rashcream. In some embodiments, MSM inhibits contamination whilesimultaneously conferring a beneficial cosmetic effect or healthbenefit.

In several embodiments, MSM is added to medicinal products or equipmentsusceptible to microbial contamination. Medicinal products include, butare not limited to, cold or flu preventatives and treatment, allergypreventatives and treatment, nasal irrigants, medicinal drops, eyedrops, inhalants, athletes foot treatments, herpes and cold soremedications, burn creams, ointments for cuts and infection, andbactericidal, fungicidal and virucidal sprays or lotions. In someembodiments, MSM is used to inhibit microbial activity on inhalers,nebulizers, ventilators, catheters, syringes, intubation tubes, hospitalroom equipment, furniture and surfaces, diagnostic equipment, fabric,bedding, and patient coverings. In several embodiments, MSM is used todisinfect bodily tissues and fluids. For example, MSM may be used aspart of a dialysis system to inhibit microbial activity in blood, whichmay be particularly helpful for sepsis patients. In another embodiment,MSM is injected into a patient to inhibit microbial activity locally orsystemically. In other embodiments, a composition including MSM istopically applied to a microbial infection present on a dermal surface.

In some embodiments, MSM products are used nasally. In otherembodiments, such products are used orally and/or as a vapor. In stillother embodiments, the product is a repeated use eye drop or otherocular medicinal product.

In several embodiments, MSM is added to medicinal products used toprevent or treat fungal infections. In some such embodiments, theproduct is used to prevent or treat athlete's foot. In some embodiments,the product is used topically. In some such embodiments, the product isa cream, ointment, spray, gel or powder. In other embodiments, theproduct is used orally.

In several embodiments, MSM inhibits the activity of mycotoxins, toxicmetabolites produced by an organism of the fungus kingdom, includingmushrooms, molds, and yeasts. Products comprising MSM are also usefulfor decontaminating surfaces and equipment that are susceptible tocontamination by such organisms and/or metabolites. In some embodiments,MSM inhibits the activity of organisms of the fungus kingdom (e.g.,mushrooms, molds, and yeasts). In yet other embodiments, MSM inhibitsthe toxins from microbes directly and/or indirectly by inhibiting theactivity of the microbes. In one embodiment, MSM inhibits the formationand/or release of microbial metabolites.

In several embodiments, MSM is added to products used to prevent ortreat viral infections. Anti-viral nasal sprays comprising MSM areprovided in one embodiment. Products comprising MSM are also useful fordecontaminating surfaces and equipment that are susceptible to viralcontamination. In one embodiment, MSM is used to inhibit the flu virus,including H1N1, either in a biological tissue or on an external surface.In some embodiments, MSM is used to inhibit the human immunodeficiencyvirus, herpes simplex virus, papilloma virus, parainfluenza virus,influenza, hepatitis, and other viruses.

In some embodiments, MSM inhibits algae. In one embodiment, MSM inhibitsalgal blooms. In some embodiments, MSM inhibits undesired phytoplanktonactivity. In other embodiments, MSM inhibits macroalgal species. Inanother embodiment, MSM inhibits dinoflagellates of genus Alexandriumand Karenia. In several embodiments, MSM inhibits the toxic metabolites(including by-products) of algae.

In some embodiments, MSM is added to medicinal products used to treat aburn, cut or wound. Wounds may include, but are not limited to,lacerations, split lacerations, over stretching, grinding compression,cut lacerations, tearing, incisions, incised wounds, abrasions, puncturewounds, penetration wounds. In some embodiments, MSM is incorporatedinto a bandage used to cover a wound. In other embodiments, MSM is addedto a cream or ointment. In some such embodiments, the product formulatedwith MSM acts an antiseptic.

Skin microflora (bacteria, fungi, viruses, phage, archaea) play asignificant role in common dermatological conditions, such as atopicdermatitis (a common form of eczema). Typically, a specific microbecolonizes the skin to disrupt the balance of commensal microflora, ormicrobes release toxic substances or invade cells to induce aninflammatory response directly. Thus, in some embodiments, MSM isincorporated into a topical product that inhibits growth of suchmicroflora. Subcutaneous administration of MSM is provided in otherembodiments.

In other embodiments, MSM is added to optical products susceptible tomicrobial contamination and/or to optical products to enhance theirantimicrobial activity. Optical products may include solutions forcleaning or disinfecting contact lenses. In some such embodiments, MSMis incorporated into various products applied to contact lenses such asa contact lens storage solution. In some embodiments, MSM is added toeye drops used in conjunction with contact lenses. In other embodiments,MSM is added to chemical solutions used in ocular diagnostic procedures,such as a multi-use pupillary dilation solution.

In several embodiments, MSM is added to oral products susceptible tomicrobial contamination and/or to oral products to enhance theirantimicrobial activity. In some embodiments, such products are used fortooth cleaning. In some embodiments, MSM is incorporated into toothpasteor tooth-gel. In some embodiments, MSM is incorporated into or coatedonto the bristles of a toothbrush. In other embodiments, MSMformulations are incorporated into or used to coat tooth floss. In otherembodiments, the product is used for cleaning the tongue. In otherembodiments, the product is a mouthwash, mouth-rinse or mouth-irrigantfor at home or professional dental use. In yet other embodiments, theproduct is a gum or confectionary. In some embodiments, MSM is added tostorage or cleaning solutions for dental implants, dentures, and thelike.

In some embodiments, MSM is added to foods containing probioticorganisms, such as milk, yogurt, rice yogurt, frozen yogurt, kefir,juice, pickled vegetables, fermented cabbage, fermented bean paste,brined olives, chocolate, cheeses and other dairy products, and certaincereals. In some embodiments, MSM is added to products that are dietarysupplements, including, but not limited to, probiotic pills, capsules,and liquids. In some such embodiments, MSM is added to a supplement forhuman ingestion. In other embodiments, MSM is added to a supplement foranimals. In some embodiments, MSM is added to a product that isformulated as a capsule or tablet. In some embodiments, MSM is added toa product that is formulated as a solid or liquid. In other embodiments,MSM is added to a foodstuff during the production process, while instill other embodiments, MSM is added to finished food products.

In several embodiments, MSM is added to a food product that issusceptible to microbial infection. In some embodiments, MSM may bemixed, ad-mixed, compounded, or otherwise incorporated into the foodproduct. In other embodiments, MSM is applied to the surface of a foodproduct. For example, in some embodiments, MSM may be sprayed on a foodproduct. Such food products may include, but are not limited to, fruits,vegetables, fish and meat products. In some embodiments, MSM is used inprocessing or packing facilities to extend the shelf-life of foodproducts. In several embodiments, the addition of MSM (e.g., about 5% toabout 25%) increases the time to spoilage of ingestible products. Forexample, MSM can be baked into or added to breads, pastries, or dough toincrease the shelf life of the edible products by about 10% to 100%(e.g., 20%, 30%, 40%, 50%, 75%, 150%, 200% or more). For example, in oneembodiment, if the shelf life of an edible product is 10 days, theaddition of MSM will increase the shelf life to at least 11 days in someembodiments (e.g., 11 days, 14 days, 15 days, 20 days, or 25 days). As afurther example, in another embodiment, if an edible product has a shelflife of 14 days at room temperature and/or 30 days in the refrigeratorand/or 3 months in the freezer, the addition of MSM will increase theshelf life to 30 days at room temperature and/or 60 days in therefrigerator and/or 6 months in the freezer. In further embodiments, theuse of MSM will permit the shipping and/or storage of an edible productat room temperature, where the product would otherwise have to beshipped and/or stored at colder temperatures. In yet other embodiments,the use of MSM will obviate the need for sterilization of edibleproducts.

In some examples, any of the disclosed compositions of MSM consistessentially of water. For example, MSM is particularly effective whencombined with water or other liquid components. In some examples, adisclosed composition of MSM is bleach-free, alcohol free or acombination thereof. In several embodiments, a composition formodulating microbial activity includes a MSM related compound instead ofor in addition to MSM. Related compounds include, but are not limitedto, DMSO and dimethylsulfide (DMS).

MSM used according to any of the embodiments provided herein may beisolated, purified or processed. MSM that is designated GRAS (GenerallyRecognized As Safe) is used for several embodiments described herein.Formulations for consumption by humans, domesticated animals andlivestock are provided in accordance with several embodiments herein.

In some embodiments, MSM is combined with one or more of the followingingredients (or derivatives, metabolites, precursors, oils, extracts,esters, acids, salts, and related compounds thereof): abietic acid,acacia, acacia senegal gum, acai extract, acetic acid, acetone, acetylglycosamine, acmella oleracea extract, adenophora stricta, alariamarginata (sea vegetable), albumin, alcohol, aldenine, alfalfa, algaeextract, alkyl guanine transferase, alkyloamides, allantoin, aluminumhydroxide, almond, aloe vera, alpha lipoic acid, aluminum benzoate,aluminum chloride, amino acids, aminopropane sulfonic acid 3, ammoniumglycolate, ammonium lauryl sulfate, anemarrhenae asphodeloides rootextract, anise oil, antioxidants, apigenin, apricot, apricot kernel,arachidonic acid, arbutin, argan oil, argania spinosa leaf extract,arginine, argireline, arnica extract, artemisia dracunculus (tarragon)oil, ascorbic acid, ascorbyl palmitate, ascorbyl tetraisopalmitate,aspergillus ferment, aspidosperma quebracho, astaxanthin, atelocollagen,avena sativa (oat) kernel extract, avobenzone, azelic acid, azuki beans,balm mint extract, balsam peru, bamboo stem extract, barely (hordeumvulgare) extract, barium sulfate, barley, basil, bee pollen, beeswax,bentonite, benzoyl peroxide, beta vulgaris root extract (beet),betacarotene, bilberry, biotin, bismuth oxychloride, bladderwrackextract, borage oil, boric acid, boric oxide, bovine placenta liquid,brewers yeast, bronopol, butyl acetate, butyl stearate, butylatedhydroxyanisole, butylated hydroxytoluene, butylene glycol, butylparaben,butyrospermum parkii, c18-36 acid triglyceride, caffeine, calamine,calcium, calendula extract, carnauba wax, camellia oleifera leafextract, camellia sinenis leaf extract, camphor, canaga odorata (ylangylang) flower oil, candelilla cera, candelilla wax, canola sterols,caprylic acid, caprylic/capric triglyceride, caprylyl glycol, caprylylglycol, capsicum oleoresin, caramel, carmine, carotenoids, carrageenan,carrot oil, carrot seed oil, carthamus tinctorius (safflower) seed oil,castor oil, cellulose, centella asiatica, calendula officinalis, ceraalba, cera carnauba, ceramide, cerebrosides, cerric ammoniumferrocyanide, ceteareth-3, cetearyl alcohol, cetearyl glucoside,cetearyl olivate, cetyl alcohol, cetyl lactate, chamomile oil,chamomilla recutita (matricaria) flower extract, chestnut, chestnutextract, chloroxylenol, chlorphenesin, cholesterol, choline, chondruscrispus (irish moss), chromium hydroxide green, chromium oxide greens,cinnamyl alcohol, citric acid, citronellol, citrus, citrus nobilis(green mandarin) oil, clove powder, clover blossom extract, clycerylcoconate, cocamidopropyl betaine, cocoa, cocoa butter,caprylate/caprate, coconut oil, coconut wax, cod liver oil, coenzymeq10, collagen, comfrey extract, coneflower extract, copernica cerifera(carnauba) wax, copper, coriander, coriandrum sativum (cilantro) oil,corn starch, cornflower extract, creatine, crithmum maritimum extract,cucumber, cyclomethicone, cyclopentasiloxane, dantoin 685, decylglucoside, deionized water, diazolidinyl urea, dicalcium phosphatedehydrate, dicaprylyl carbonate, diethanolamine, dilaurate, dimethicone,dimethylaminoethanol, dioscorea villosa (wild yam) root extract,dipotassium glycyrrhizinate, disodium distyrylbiphenyl disulfonate,disodium edta, dmdm hydantoin, echinacea angustifolia (coneflower)extract, edta, eijitsu rose, elaeis oleifera, elastin, elder flower,emollients, enzymes, epilobium fleischeri extract (gravel willow),equisetum hiemale leaf extract (horsetail), erucate, essential fattyacids, essential oils, ethanol, ethoxydiglycol, ethyl acetate,ethylene/acrylic acid copolymer, ethylhexyl palmitate,ethylhexylglycerin, ethylparaben, eucalyptus extract, eukarion, euterpeoleracea fruit extract, evening primrose oil, exfoliants, fatty acids,fatty alcohols, fennel oil, ferric oxide, flavanoids, flavonolignan,fish oils, flax, floralozone, fluoride, formaldehyde, fruit acids, fruitextract, fruit extracts, gaba, gamma linolenic acid, gelatin, geraniol,geranium oil, gigartina papillata (wildcrafted seaweed), ginger, gingeroil, ginko biloba oil, ginseng, glucosamine, glucose oxidase, glucosesugar, glycereth, glycereth-26, glycerin, glycerol, glycerol stearate,glyceryl hydrogenated rosinate, glyceryl oleate, glyceryl stearate,glycol distearate, glycolic acid, gold, goldenseal extract, grape seed,grape seed oil, grapefruit, grapefruit oil, grapefruit seed extract,green tea, gums, hazelnut oil, hdi/trimethylol hexyllactonecrosspolymer, hemp seed oil, hexamidine, hexylene glycol, homosalate,honey, hordeum distychum extract, hordihydroguaiaretic acid, hormones,humectant, humulus lupulus extract, hyaluronic acid, hydrastiscanadensis extract (golden seal), hydrocortisone, hydrocotyl extract,hydrogenated castor oil laurate, hydrogenated polyisobutene,hydrogenated polysobulene, hydrolyzed animal protein, hydrolyzedkeratin, hydrolyzed rhizobian gum, hydrolyzed soy protein, hydrolyzedwheat protein, hydroxy acids, hydroxyethylcellulose,hydroxyethyl-cellulose, hydroxyisohexyl 3-cyclohexene carboxaldehyde,hydroxypropyl-cellulose, hydroxypropyltrimonium honey, hydroquinone,hydroxystearate, hypericum extract, idebenone, imidazolidinyl urea,iodine, irish moss, iron oxides, isobutylparaben, isododecane,isohexadecane, isononyl isononanoate, isopentyldiol, isopropyl alcohol,isopropyl lanolate, isopropyl linoleate, isopropyl myristate,isostearate, isostearic acid, ivy extract, jasmine oil, jojoba butter,jojoba oil, juniper extract, juniper oil, kaolin, keratin, ketones,kinerase, kinetin, kojic acid, kukui nut oil, lactic acid,lactoperoxidase, lady's mantle leaf extract, laminaria digitata (kelp)extract, lanolin, larix sibirica wood extract, lauramide, laurate,laureth, lauryl alcohol, lauryl glucoside, lavender, lavender oil,lecithin, lemon oil, licorice, lime oil, limonene, linden extract,linoleic acid, linolenic acid, liposomes, locust bean, lycium barbarumfruit extract, lycium barbarum fruit extract (goji berry), lycopene,macadamia nut oil, matcha, magnesium aluminum silicate, magnesiumascorbyl phosphate, magnesium myristate, magnesium stearate, magnesiumsulfate (epsom salts), malpighia punicifolia (acerola) fruit extract,manganese violet, mango butter, marigold, marshmallow extract, matchatea powder, matricaria oil, mea, meadowsweet, melaleuca oil, melonextract, mentha piperita (organic peppermint) extract, menthol, methylacetate, methyl ethyl ketone, methyldihydrojasmonate, methylparaben,mica, microdermabrasion compounds, milk protein, minerals, mineral oil,mipa, monoethanolamine, monostearate, montmorillonite (green clay),mugwort (artesemia vulgaris) extract, mulberry, mulberry (morus nigra)root extract, murumuru, mushrooms, myristate, myristate, myristic acid,myristyl myristate, myrtus communis (green myrtle) oil, n-acetylglucosamine, nephrite powder, neroli oil, nettle leaf, neuropeptides,niacin, nitrosamines, nonyl nonoxynol-150, nucleic acids, nutmeg powder,nuts, oat, oatmeal, oats, ocimum basilicum linalol (basil linalol) oil,octinoxate, octsalate, oleate, oleic acid, oleyl alcohol, oligopeptides,oligosaccharides, olive fruit extract, olive oil, omega-3, orange peeloil, orthoboric acid, oxybenzone, ozokerite, padina pavonica thallusextract, palm oil, palmitate, palmitic acid, palmitoyl, pantethine,panthenol, para-aminobenzoic acid, paraben, paraffin, passifloraincarnata fruit extract, passionfruit, patchouli, peach stone, peatextract, pectin, peg, peppermint, peppermint oil, peptides, petroleumjelly, phellodendron amurense bark extract, phenoxyethanol, phenyltrimethicone, phenylethyl resorcinol, phosphoric acid, phytochemicals,pine tree extract derivative, pineapple extract, plantago lanceolataleaf extract, plantain leaf extract, pollen extract, polygonumcuspidatum root extract, polypeptides, polysaccharides, polysilicone,polysorbate, polysorbate, polyvinylpyrrolidone, progesterone, propyleneglycol, propylheptyl caprylate, propylparaben, pumpkin seed extract,punica granatum (pomegranate) extract, punica granatum extract/punicagranatum, pycnogenol, quaternium-15, quillaja saponaria (soap) barkextract, quillia extract, reserveratol, retinoic acid, retinoids,retinol, retinyl palmate, ribes rubrum fruit extract, rice, rice branwax, ricinoleate, rose oil, rosehips, rosemary, rosemary oil, rosewater,royal jelly, rubus villosus fruit extract, saccharum officinarum (canesugar), salicylic acid, salvia, sandalwood oil, saponins, sassafras, sawpalmetto, saxifraga sarmentose extract, sclareolide, scutellariabaicalensis extract, seaweed, secale cereale (rye) seed extract,selaginella tamariscina (spike moss) extract, selenium, sesame oil,sesquioleate, shavegrass herb, shea butter, silibinin, silica, silicone,sirtuins, sodium alginate, sodium ascorbate, sodium bisulphate, sodiumborate, sodium carbonate, sodium chloride, sodium citrate, sodiumdehydroaceatate, sodium ethylparaben, sodium glycyrrhetinate, sodiumhyaluronate, sodium lactobionate, sodium lauryl sulfate, sodiummethylparaben, sodium polystyrene sulfonate, sodium propylparaben,sodium stearate, sodium thioglycolate, sodium acrylodimethyl taurate,sorbitan isostearate, sorbitan olivate, sorbitan sesquioleate, sorbitanstearate, sorbitol, sorbitol, soy, soy wax, soybean oil, spearmint oil,squalane, st. paul's/john's wort, stearate, stem cells, sucrosestearate, sugar cane extract, sulfate, sunflower seed oil, sweet almondoil, symphytum officinale (comfrey) leaf extract, symphytum officinaleleaf extract, synthetic fluorphlolopite, tamarindus indica seed extract,tea tree oil, thyme extract, tin oxide, titanium dioxide, titaniumdioxide, tocopherol, tocopheryl acetate, tocopheryl acetate, toluene,tomato, tragacanth, tretinoin, tribehenin, triclosan, tridecyltrimellitate, triethanolamine, trihydroxystearin, triisostearyl citrate,trimethylolpropane triisostearate, trimethylsiloxysilicate,trimyristate, tripeptide, turmeric, tyrosine, ubiquinone, ultramarines,undecylenoyl phenylalanine, urea, uridine, vaccinium macrocarpon fruitextract, vegetable glycerin, vetiver oil, vitamin A, vitamin B1-B12,vitamin C, vitamin C ester, vitamin D, vitamin E, vitamin K, vitamins,walnut shell powder, water, wheat germ oil, whey protein (lactisproteinum), white birch bark extract, willowbark, wintergreen oil, witchhazel, xantham gum, xanthan gum, yarrow extract, yeast, yerba mate,yucca, zinc oxide, zinc stearate.

In some embodiments, the composition comprises, consists or consistsessentially of MSM in combination with one, two, three, four, five ormore of the above-identified ingredients. In several embodiments, MSMinhibits microbial activity in the formulation. In other embodiments,MSM offers the same or better antimicrobial effect when used to replacea preservative in the formulation (some of which are identified above).In certain embodiments, MSM offers the same or better antimicrobialeffect when used with a reduced amount of preservative. In yet otherembodiments, the addition of MSM to a formulation having a preservativeenhances the effects of the preservative. The ingredients identifiedherein may be used with MSM in a cosmetic formulation (e.g., oral,injectable, or topical), or in other types of formulations (e.g., oral,injectable, or topical medical formulations).

In some embodiments, the composition includes MSM, but is free of one ormore of the following compounds: sulfates, GMOs, synthetic fragrances,synthetic dyes, formaldehyde, potassium sorbate, methylparaben,methylchloroisothiazolinone, cocamidopropyl betain, parabens, decylpolyglucose, polyaminopropyl biguanide, phenoxyethanol, sodium laurethsulfate, tetrasodium EDTA, decyl glucoside, polyethylene glycol,propylene glycol, phthalates, and triclosan. In some embodiments, theuse of MSM permits the manufacture of a formulation that is free of anysynthetic ingredient. In yet other embodiments, the use of MSM permitsthe manufacture of a formulation that is free of any allergy-causing,immunosuppressant and/or inflammatory ingredient.

In several embodiments, the antimicrobial properties of MSM reduce oreliminate the need for sterilization, lowered temperatures, sterileenvironments, special closures, and/or special packaging, etc. MSM has adual or multipurpose function according to some embodiments. Forexample, not only does MSM inhibit the growth of undesiredmicroorganisms, MSM also beneficially affects the product to which it isadded in several embodiments (e.g., MSM serves as an antioxidant,regenerative compound, anti-wrinkle compound, moisturizer, skinbrightener, softener, circulation stimulant, neutralizer, reparative,hair/nail strengthener, healing catalyst, resurfacing agent, etc., orcombinations of two or more thereof). In several embodiments, theantimicrobial properties of MSM increase the shelf-life, half-life,efficacy and/or stability of the formulation (or specific ingredientidentified herein). The use of MSM may be particularly beneficial insome embodiments for cosmetic or other types of formulations that areshared by more than one person (e.g., cosmetics used by make-up artistsor at cosmetic counters).

Cosmetics may include, but are not limited to, lipstick, lip gloss, lipliner, lip plumper, lip balm, lip conditioner and lip boosters,foundation, powder, rouge, blush, bronzer, mascara, eye liner, eyeshadow, eye shimmer, glitter eye pencils, eyebrow pencil, nail polish,concealer, skin care products (e.g., microdermabrasion products,soothing gels) creams, lotions, serums, moisturizer, sunscreen, skinrepair products (e.g., for acne, sunburn, wrinkles, dark circles), andscrubs. Face, hair, and body formulations (e.g., shampoo, soaps,conditioners, sprays, gels, serums, restorative treatments, deodorants,etc.) are provided in several embodiments. Cosmetics, such ascosmeceutical and nutraceutical products, are provided in severalembodiments of the invention. Dermal fillers and other dermatologicalproducts (such as hyaluronic acid, waglerin 1, acetyl hexapeptide-8,palmitoyl tetrapeptide-7, palmitoyl oligopeptide, liposomes, collagen,calcium hydroxyl-apatite, poly-lactic acid, and botulinum toxin) areprovided in several embodiments. Anti-wrinkle, anti-acne, anti-aging,exfoliating, moisturizing, and anti-stretch mark formulations,fragrances, mineral make-up, and primers are provided in severalembodiments. Dermal gels, for cosmetic and medical use (e.g., inhibitingor preventing microbial infection and/or wound healing) are provided insome embodiments.

In several embodiments, products containing MSM can be shipped and/orstored in high temperature and high humidly conditions that wouldotherwise be favorable for microbial activity.

In some embodiments, products including MSM are packaged in containersadapted for multiple use applications and exposure to externalmicroorganisms, such as from the air or contact with a body part (e.g.,fingers). The use of MSM is particularly beneficial in severalembodiments because it increases the shelf life of such products. Insome embodiments, products comprising MSM are also packaged insingle-use sealed containers. In one embodiment, a single-use product(such as a condiment packet, seasoning packet, a travel cosmeticpackage, etc.) will have a longer shelf life and/or will no longer needrefrigeration if MSM is used in conjunction with the product and/orpackaging.

In some embodiments, MSM is incorporated directly into packagingmaterials to, for example, enhance shelf life. For example, MSM may beincorporate into food storage bags to inhibit microbial growth. In otherembodiments, MSM may be incorporated into containers and/or lids toenhance shelf life of foods, cosmetics and other products by inhibitingundesired microbial growth. In yet other embodiments, MSM may beincorporated into liner products such as plastic and cling wraps.

In several embodiments, a composition for inhibiting microbial activityin a cream or topical ointment includes MSM, wherein the MSM isconfigured for affecting the microbial contamination by inhibitingmicrobial activity. MSM is provided in a concentration of at least 5%according to one embodiment (e.g., 5-10%, 10-16%, 16-20%, 20-30%,30-40%, 40-50%, 50-75% or higher, and overlapping ranges thereof). Insome examples, the composition is a preservative-free cream. In oneembodiment, MSM inhibits microbial activity by at least 50% in the creamat room temperature.

In some embodiments, pharmaceutical compositions include MSM, DMSO,and/or antimicrobial agents, or combinations thereof, which areformulated for use in human or veterinary medicine.

For example, the provided pharmaceutical compositions include about0.01% MSM by weight to about 20% MSM by weight. In some embodiments, apharmaceutical composition contains between about 0.01% to about 5% MSMby weight. Other embodiments contain between about 5% to about 10% MSM,about 10% to about 15% MSM, or about 15% to about 20% MSM, such as about5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%,about 19% or about 20% MSM. Some embodiments include about 10-16% MSM,about 10-14% MSM or about 10-12% MSM.

Exemplary additional anti-microbial agents which can be included in adisclosed composition include, but are not limited to penicillinderivatives, cephalosporins, penems, monobactams, carbapenems,Beta-lactamase inhibitors and combinations thereof. Examples ofpenicillin derivatives include, but are not limited to, aminopenicillins(e.g., amoxacillin, ampicillin, and epicillin); carboxypenicillins(e.g., carbenicillin, ticarcillin, and temocillin); ureidopenicillins(e.g., azlocillin, piperacillin and mezlocillin); mecillinam,sulbenicillin, benzathine penicillin, penicillin G (benzylpenicillin),penicillin V (phenoxymethylpenicillin), penicillin O(allylmercaptomethylpenicillinic), procaine penicillin, oxacillin,methicillin, nafcillin, cloxacillin, dicloxacillin, flucloxacillin,pivampicillin, hetacillin, becampicillin, metampicillin, talampicillin,co-amoxiclav (amoxacillin plus clavulanic acid), and piperacillion.Examples of cephalosporins include, but are not limited to, cephalexin,cephalothin, cefazolin, cefaclor, cefuroxime, cefamandole, cefotetan,cefoxitin, ceforanide, ceftriaxone, cefotaxime, cefpodoxime proxetil,ceftazidime, cefepime, cefoperazone, ceftizoxime, cefixime andcefpirome. Examples of penems include, without limitation, faropenem.Examples of monobactams include, without limitation, aztreonam andtigemonam. Examples of carbapenems include, but are not limited to,biapenenvdoripenem, ertapenem, -imipenem, -meropenem, -and panipenem.Examples of Beta-lactamase inhibitors include, but are not limited to,tazobactam([2S-(2alpha,3beta,5alpha)]-3-Methyl-7-oxo-3-(1H-1,2,3-triazol-1-ylmethyl)-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylicacid 4,4-dioxide sodium salt), sulbactam(2S,5R)-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylicacid 4,4-dioxide sodium), and clavulanic acid((2R,5R,Z)-3-(2-hydroxyethylidene)-7-oxo-4-oxa-1-aza-bicyclo[3.2.0]heptane-2-carboxylicacid), or other Beta-lactam antibiotic.

Many antibiotics have an established minimum inhibitory concentration(MIC) at which they are effective in reducing or killing certainbacteria. In some embodiments, a disclosed pharmaceutical compositionincludes an amount of Beta-lactam antibiotic equal to about 0.001 to 100MIC for the particular bacterial pathogens disclosed herein. In someembodiments the pharmaceutical composition comprises about 1-5, 5-10,10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90 or about 90-100MIC of a Beta-lactam antibiotic. In some embodiments, the pharmaceuticalcomposition comprises about 0.001, 0.01, 0.1, 0.5 or 1 MIC of aBeta-lactam antibiotic.

Pharmaceutical compositions provided herein also include combinations ofMSM and antimicrobial compounds, for example a combination of MSM and aBeta-lactam antibiotic. In some embodiments, pharmaceutical compositionsprovided herein include 10-16% MSM and an amount of a Beta-lactamantibiotic equal to 1 MIC for a bacterial pathogen the composition willbe contacting.

One of skill in the art will know the MIC of an antibiotic for aparticular bacterial pathogen, or the skilled artisan will know how todetermine the MIC of an antibiotic for a particular bacterial pathogen.Methods of determining a MIC of a particular antibiotic for a particularbacterial pathogen are disclosed herein, for example use of the Etest®antibiotic testing system (bioMérieux, Durham, N.C.).

The dosage form of the pharmaceutical composition will be influenced bythe mode of administration chosen. For instance, in addition toinjectable fluids, inhalational, topical, ophthalmic, peritoneal, andoral formulations can be employed. Inhalational preparations can includeaerosols, particulates, and the like. In general, the goal for particlesize for inhalation is about 1 μm or less in order that thepharmaceutical reach the alveolar region of the lung for absorption.

Pharmaceutical compositions that include MSM, DMSO, an antimicrobialagent or therapeutic compound as described herein such as an activeingredient, or which include a mixture of two or more thereof, with orwithout additional agent(s) as active ingredients, may be formulatedwith an appropriate solid or liquid carrier, depending upon theparticular mode of administration chosen. Oral formulations may beliquid (for example, syrups, solutions, or suspensions), or solid (forexample, powders, pills, tablets, or capsules). For solid compositions,conventional non-toxic solid carriers can include pharmaceutical gradesof mannitol, lactose, starch, or magnesium stearate. Actual methods ofpreparing such dosage forms are known, or will be apparent, to those ofordinary skill in the art.

For oral administration, the pharmaceutical compositions can take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents (forexample, pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (for example, lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(for example, magnesium stearate, talc or silica); disintegrants (forexample, potato starch or sodium starch glycolate); or wetting agents(for example, sodium lauryl sulphate). The tablets can be coated bymethods well known in the art. Liquid preparations for oraladministration can take the form of, for example, solutions, syrups orsuspensions, or they can be presented as a dry product for constitutionwith water or other suitable vehicle before use. Such liquidpreparations can be prepared by conventional means with pharmaceuticallyacceptable additives such as suspending agents (for example, sorbitolsyrup, cellulose derivatives or hydrogenated edible fats); emulsifyingagents (for example, lecithin or acacia); non-aqueous vehicles (forexample, almond oil, oily esters, ethyl alcohol or fractionatedvegetable oils); and preservatives (for example, methyl orpropyl-p-hydroxybenzoates or sorbic acid). The preparations can alsocontain buffer salts, flavoring, coloring, and sweetening agents asappropriate.

For administration by inhalation, the compounds for use according to thepresent disclosure are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, for example, dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit can be determined by providing a valve to deliver a metered amount.Capsules and cartridges for use in an inhaler or insufflator can beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

For topical administration, the compounds can be, for example, mixedwith a liquid delivery agent for administration locally. The agents usedtherapeutically (such as DMSO, MSM and/or other therapeutic compounds asdescribed herein) are readily soluble or suspendable in water, and assuch this would be useful for delivery since water does not causeadverse biological tissue effects. This allows sufficiently high dosesto be administered locally or systemically, without secondary toxicityfrom the delivery vehicle.

Pharmaceutical compositions that include a therapeutic amount of MSM asdescribed herein as an active ingredient will normally be formulatedwith an appropriate solid or liquid carrier, depending upon theparticular mode of administration chosen. The pharmaceuticallyacceptable carriers and excipients useful in this disclosure areconventional. For instance, parenteral formulations usually compriseinjectable fluids that are pharmaceutically and physiologicallyacceptable fluid vehicles such as water, physiological saline, otherbalanced salt solutions, aqueous dextrose, glycerol or the like.Excipients that can be included are, for instance, proteins, such ashuman serum albumin or plasma preparations. If desired, thepharmaceutical composition to be administered may also contain minoramounts of non-toxic auxiliary substances, such as wetting oremulsifying agents, preservatives, and pH buffering agents and the like,for example sodium acetate or sorbitan monolaurate. Actual methods ofpreparing such dosage forms are known, or will be apparent, to thoseskilled in the art.

The pharmaceutical compositions that include a therapeutic effectiveamount of MSM, in some embodiments, will be formulated in unit dosageform, suitable for individual administration of precise dosages. Theamount of MSM administered will be dependent on the subject beingtreated, the severity of the affliction, and the manner ofadministration, and is best left to the judgment of the prescribingclinician. Within these bounds, the formulation to be administered willcontain a quantity of the active component(s) in amounts effective toachieve the desired effect in the subject being treated.

Preparations for administration can be suitably formulated to givecontrolled release of the therapeutic agent(s) (e.g., DMSO, MSM,Beta-lactam antibiotic and so forth). For example, the pharmaceuticalcompositions may be in the form of particles comprising a biodegradablepolymer and/or a polysaccharide jellifying and/or bioadhesive polymer,an amphiphilic polymer, an agent modifying the interface properties ofthe particles and a pharmacologically active substance. Thesecompositions exhibit certain biocompatibility features that allow acontrolled release of the active substance. See, for example, U.S. Pat.No. 5,700,486.

Polymers can be used for controlled release. Various degradable andnondegradable polymeric matrices for use in controlled drug delivery areknown in the art (Langer, Accounts Chem. Res. 26:537, 1993). Forexample, the block copolymer, polaxamer 407 exists as a viscous yetmobile liquid at low temperatures but forms a semisolid gel at bodytemperature. It has shown to be an effective vehicle for formulation andsustained delivery of recombinant interleukin-2 and urease (Johnston etal., Pharm. Res. 9:425, 1992; Pec, J. Parent. Sci. Tech. 44(2):58,1990). Alternatively, hydroxyapatite has been used as a microcarrier forcontrolled release of proteins (Ijntema et al., Int. J. Pharm. 112:215,1994). In yet another aspect, liposomes are used for controlled releaseas well as drug targeting of lipid-capsulated compounds (Betageri etal., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc.,Lancaster, Pa., 1993). Numerous additional systems for controlleddelivery of therapeutic proteins are known (e.g., U.S. Pat. No.5,055,303; U.S. Pat. No. 5,188,837; U.S. Pat. No. 4,235,871; U.S. Pat.No. 4,501,728; U.S. Pat. No. 4,837,028; U.S. Pat. No. 4,957,735; andU.S. Pat. No. 5,019,369; U.S. Pat. No. 5,055,303; U.S. Pat. No.5,514,670; U.S. Pat. No. 5,413,797; U.S. Pat. No. 5,268,164; U.S. Pat.No. 5,004,697; U.S. Pat. No. 4,902,505; U.S. Pat. No. 5,506,206; U.S.Pat. No. 5,271,961; U.S. Pat. No. 5,254,342; and U.S. Pat. No.5,534,496).

In several embodiments, pharmaceutical compositions include DMSO and/orMSM, and a therapeutic agent to treat an infectious disease, such asH1N1, herpes simplex virus, or HIV. In some embodiments, compositionsincluding DMSO and/or MSM are provided as an inhalant to treat aninfectious disease. In some embodiments, pharmaceutical compositions fortreating an infectious disease include DMSO and/or MSM formulated assolids, while in several other embodiments, compositions including DMSOand MSM are formulated as liquids. In some embodiments, the compositionsare consumed orally to treat the infectious disease, while in some otherembodiments, the compositions are applied topically. In one particularembodiment, the compositions are delivered in an inhalant device whichis configured to generate particles of the formulation that range insize from about 0.5 um to about 5 um.

In some embodiments, the pharmaceutical compositions including DMSOand/or MSM allow antibiotics (or other therapeutic agents) to penetratelung tissue infected with an infectious disease. In one embodiment, suchcompositions including DMSO and/or MSM: (i) allow antibiotics to reachdeeper levels of infected tissue; (ii) allow direct contact of infectedtissue; (iii) lengthen the exposure time of the antibiotic to theinfected tissue; and/or (iv) decrease the time to achieve a desiredantibiotic effect. In one embodiment, DMSO and/or MSM achieves one ormore of these desired effects through use as an inhalant, wherein theinhalant additionally comprises one or more antibiotics or othertherapeutic agents.

In some embodiments, pharmaceutical compositions including DMSO and/orMSM formulations further include antiparasitic agents that are effectivein treating infections caused by parasites, such as nematodes, cestodes,trematodes, protozoa, or amoebae.

In some embodiments, pharmaceutical compositions including DMSO and/orMSM formulations further include antifungal agents that are effective intreating fungal infections, such as those caused by ringworm,candidiasis, and Cryptococcus (cryptococcal meningitis, for example).

In some embodiments, pharmaceutical compositions including DMSO and/orMSM formulations further include antiviral agents that are effective intreating viral infections. In some embodiments, specific classes ofantiviral agents are used to treat infections caused by a particulartype of virus. In some embodiments, agents that target HIV, herpesviruses, hepatitis B or C viruses, and influenza viruses, such as H1N1,are used.

In several embodiments, DMSO and/or MSM compositions include antibioticsthat are effective in treating bacterial infections by, for example,inhibiting bacterial growth, metabolism, proliferation, activity and/orfunction. In some embodiments, bacteriostatic antibiotics are used,while in other embodiments, bactericidal antibiotics are used. In stillother embodiments, both bacteriostatic and bactericidal antibiotics areincorporated into a single formulation comprising DMSO and/or MSM. Insome embodiments, antibiotics of one or more classes are incorporatedinto a composition including DMSO and/or MSM. In certain embodiments, acomposition includes one or more of an: aminoglycoside, ansamycin,carbacephem, carbapenem, cephalosporin (1^(st), 2^(nd), 3^(rd), 4^(th),or 5^(th) generation), glycopeptides, macrolide, monobactam, penicillin,polypeptide, quinolone, sulfonamide, tetracycline, and the like.

In some embodiments, specific diseases are targeted by incorporatingspecific antibiotics into a disclosed composition including DMSO and/orMSM. For example, macrolides, such as azithromycin or erythromycin areincorporated into formulations used to treat respiratory or mycoplasmalinfections. Likewise, penicillins, such as amoxicillin or oxacillin areincorporated into formulations used to treat a broad range ofstreptococcal infections.

In still other embodiments, specific disease-causing microorganisms aretargeted by the specific antibiotics incorporated into a formulationcomprising DMSO and/or MSM. For example, aminoglycosides, such asneomycin are incorporated into formulations used to treat Escherichiacoli infections. In several embodiments, antibiotics typically used tocombat microbial infections are used. In certain embodiments,antibiotics including, but not limited to, isoniazid, rifampicin,pyrazinamide, and ethambutol are incorporated into formulationscomprising one or more of DMSO and MSM, and are used to treat aninfectious disease, including a drug-resistant infectious disease.

In several embodiments, compositions include DMSO, MSM and one or moreof the following therapeutic agents: rifampicin, isoniazid,pyrazinamide, and ethambutol are provided. In other embodiments,compositions including DMSO and at least one of rifampicin, isoniazid,pyrazinamide, and ethambutol are provided. In further embodiments,compositions including MSM and at least one of rifampicin, isoniazid,pyrazinamide, and ethambutol are provided. In several embodiments,compositions including DMSO and/or MSM in combination with rifampicin,isoniazid, pyrazinamide, and ethambutol are provided to treat aninfectious disease, including a drug-resistant infectious disease.

In some embodiments, rifampicin is provided in a total daily doseranging from about 400 mg to about 800 mg per day. In some embodiments,rifampicin is provided in a total daily dose ranging from about 500 mgto about 700 mg per day, while in still other embodiments, it isprovided in a total daily dose ranging from about 550 to about 650 mgper day, including 560, 570, 580, 590, 600, 610, 620, 630, and 640 mgper day.

In some embodiments, isoniazid is provided in a total daily dose rangingfrom about 100 mg to about 500 mg per day. In some embodiments,isoniazid is provided in a total daily dose ranging from about 200 mg toabout 400 mg per day, while in still other embodiments, it is providedin a total daily dose ranging from about 250 mg to about 350 mg per day,including 260, 270, 280, 290, 300, 310, 320, 330, and 340 mg per day.

In some embodiments, pyrazinamide is provided in a total daily doseranging from about 1.0 to about 4.0 g per day. In some embodiments,pyrazinamide is provided in a total daily dose ranging from about 2.0 toabout 3.0 g per day, while in still other embodiments, it is provided ina total daily dose ranging from about 2.0 to 2.5 g per day, including2.1, 2.2, 2.3, and 2.4 g.

In some embodiments, ethambutol is provided in a total daily doseranging from about 0.5 to about 2.5 g per day. In some embodiments,ethambutol is provided in a total daily dose ranging from about 1.0 to2.0 g per day, while in still other embodiments, it is provided in atotal daily dose ranging from about 1.0 to about 1.5 g per day,including 1.1, 1.2, 1.3, and 1.4 g.

In some embodiments, pharmaceutical compositions including DMSO and/orMSM are used to pretreat a patient suffering from an infectious disease,such as H1N1. In some embodiments, the dose of DMSO and/or MSM used topretreat patients ranges from about 10% to 50% weight to volume. In someembodiments, the pretreatment DMSO and/or MSM dose ranges from about 20%to about 40%, from about 25% to 35%, including 26, 27, 28, 29, 30, 31,32, 33, and 34%. In some embodiments, about 50% to about 100% DMSOand/or MSM is used. In several embodiments, pretreatment with DMSOand/or MSM enhances the ability of an antibiotic to inhibit bacterialactivity and/or sensitizes a drug-resistant strain to a drug that waspreviously ineffective.

In some embodiments, a pharmaceutical composition is prepared whereinantimicrobials are dissolved in DMSO and/or MSM prior to administration.This is particularly advantageous in certain embodiments because theantimicrobial and DMSO (and optionally MSM) can be administered to asubject via inhalation. Inhalants, according to some embodiments,provide direct access of the DMSO and/or MSM to infected lung tissue tosensitize bacterial cells to the antibiotic.

In one embodiment, an inhalant is provided to target the site ofinfection (e.g., lungs) of several infectious diseases. In some suchembodiments, the inhalant device comprises a nebulizer. In otherembodiments, an inhaler is used. In some embodiments, a pressurizedmetered-dose inhaler is used, and the formulation is inhaled in liquidaerosol form. In other embodiments, dry powder inhalers are used, andthe formulation is inhaled in a powder aerosol form. In severalembodiments, oral, intravenous, intramuscular, or subcutaneousadministration is used in addition to or instead of inhalant therapy.

The ability to administer antimicrobial agents as an inhalant (e.g., ina powder aerosol form) with DMSO and/or MSM is especially advantageousin some embodiments because it allows for increased shelf-stability andpre-packaged dosages. This is particularly helpful for individuals inunderdeveloped or developing nations who do not have regular access tohealthcare facilities. Entire courses of treatment can be provided to anaffected subject in a single visit to a healthcare practitioner withoutthe need for a hospital stay or repeat visits. In several embodiments,formulations disclosed herein are suitable for self-administration(e.g., through inhalant devices) and are therefore especiallyappropriate for patients with limited access to healthcare.

In certain embodiments, the total volume of inhaled DMSO and/or MSM isabout 2-8 mL. In some embodiments, the total volume of inhaled DMSOand/or MSM is about 2 mL to about 4 mL. In some embodiments, the totalvolume of inhaled DMSO and/or MSM is about 6 mL to about 8 mL. In stillother embodiments, the total volume of inhaled DMSO and/or MSM is about3 mL to about 7 mL, including 4, 5, and 6 mL. Thus, in some embodiments,the concentration of DMSO administered via inhalation ranges from about65% to about 95%, including 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92,93, and 94%.

In several embodiments, MSM is included with the inhaled DMSO andantimicrobial compounds. In certain embodiments, the amount of inhaledMSM ranges from about 0.01% by weight to about 70% by weight of theinhalant. In other embodiments, the inhaled formulation contains betweenabout 0.01% and 10% MSM by weight. Other embodiments contain betweenabout 10 and 20% MSM, about 20-30% MSM, about 30-40% MSM, about 40-50%MSM, about 50-60% MSM, or about 60-70% MSM including 60, 61, 62, 63, 64,65, 66, 67, 68, 69, and 70% MSM. Still other embodiments comprise aformulation containing about 7 and 15% MSM, about 15-25% MSM, about25-35% MSM, about 35-45% MSM, about 55-60% MSM, about 60-65% MSM, orabout 65-70% MSM. Thus, in some embodiments of the inhaled formulationcontaining MSM, the concentration of DMSO administered ranges from about50% to about 95%, including 55, 60, 65, 70, 75, 80, 85, 86, 87, 88, 89,90, 91, 92, 93, and 94%.

In several embodiments, the use of MSM reduces the amount of DMSO neededto achieve a comparable effect and/or enhances the efficacy of DMSO byat least 10%, 25%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 50-fold,or 100-fold. In other embodiments, the use of MSM reduces the amount ofa therapeutic agent needed to achieve a comparable effect and/orenhances the efficacy of the therapeutic agent by at least 10%, 25%,50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 50-fold, or 100-fold. Infurther embodiments, the use of DMSO reduces the amount of a therapeuticagent needed to achieve a comparable effect and/or enhances the efficacyof the therapeutic agent by at least 10%, 25%, 50%, 100%, 2-fold,3-fold, 5-fold, 10-fold, 50-fold, or 100-fold. In still otherembodiments, the use of DMSO and MSM reduces the amount of a therapeuticagent needed to achieve a comparable effect and/or enhances the efficacyof the therapeutic agent by at least 10%, 25%, 50%, 100%, 2-fold,3-fold, 5-fold, 10-fold, 50-fold, or 100-fold as compared to DMSO or MSMalone and/or the therapeutic agent alone.

In several embodiments, a pretreatment formulation including DMSO, aloneor in combination with MSM, is administered to a subject intravenously,intramuscularly, topically or orally to enhance the effects of aninhalant therapy comprising DMSO and/or MSM with therapeutic agents,such as antibiotics. The pretreatment with DMSO, alone or in combinationwith MSM, enhances the inhalant's therapeutic effects by at least 10%,25%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 50-fold, or 100-fold.

In several embodiments, subjects having an infectious disease re treatedwith a formulation comprising, consisting or consisting essentially ofDMSO, alone or in combination with MSM, and one or more therapeuticagents, such as antibiotics. In some embodiments, the formulationadditionally includes other therapeutics agents, carriers or excipients.In one embodiment, the formulation additionally includes arginine,vitamin D, antioxidants, macrolides, linezolid, thioacetazone,thioridazine, or combinations thereof.

DMSO readily disrupts the integrity of many materials (particularlyplastics and polymers used in manufacturing disposable medicalequipment). Accordingly, several embodiments of the invention comprisedevices to facilitate the storage and administration of DMSO. In someembodiments, DMSO is stored in glass bottles and administered throughnon-reactive tubing. In other embodiments, inhalant devices arespecially designed to be DMSO resistant. In some embodiments, portionsof the inhalant devices are disposable or replaceable. According toseveral embodiments, formulations comprising DMSO are manufactured,stored and/or administered using materials and devices disclosed in U.S.patent application Ser. No. 12/066,480, which is the National Phaseentry of International Application No.: PCT/US06/35499, filed Sep. 11,2006, which is herein incorporated by reference in its entirety.

In certain embodiments, the delivery device delivers droplets orparticles of the inhaled formulation of a size capable of reaching thebronchioles of the patient's lungs. In some embodiments, the deliverydevice is synchronized with a patient's breathing rhythm to carry theformulation to the bronchioles. Inhalant therapy according to oneembodiment, enables more direct administration the inhaled formulationto infected pulmonary target tissues. Direct targeting is advantageousin some embodiments because it allows for reduction of the amount ofantimicrobial compounds incorporated into the formulation whilemaintaining or improving efficacy of the formulation against infectiousmicroorganisms. In other embodiments, direct administration increasesthe efficacy of a given antimicrobial regime against one or moredrug-resistant strains of microorganism. Direct targeting, according toother embodiments, minimizes side effects by minimizing contact withnon-targeted tissue.

The small droplet or particle size that is provided according to someembodiments reduces the volume of DMSO and/or MSM that is administeredas compared to traditional ventilator therapy. For example, in oneembodiment, the use of an inhalant device (e.g., nebulizer) will beefficacious with about 6 mg to about 25 mg DMSO and/or MSM daily, ascompared to 50-100 mg daily when administered through certain otherpathways. Reducing DMSO is beneficial in some embodiments because itreduces undesired side effects and odor. In other embodiments, higheramounts of DMSO are used and tolerated.

In several embodiments, the addition of MSM unexpectedly reduces theunpleasant odor normally experienced with DMSO use. For example, incertain embodiments, DMSO and MSM formulations produce no perceptibleodor after use. In some other embodiments having DMSO concentrationsapproaching or exceeding 50%, the combination with MSM in theformulation reduces or eliminates the DMSO-based odor. Such a result isunexpected, given that DMSO use is normally associated with a strongunpleasant odor.

In some embodiments, the use of DMSO and/or MSM with therapeutic agents(such as antibiotics) permits the manufacture and/or administration ofsmall droplets or particle sizes, thereby reducing the irritation of themucosa of the mouth and throat, as the droplets or particles travel moredeeply into the lungs of the patient. In some embodiments, the depth oftravel of the droplets or particles increases the concentration of thedissolved antibiotics in the patient's lungs.

In several embodiments, DMSO and/or MSM compositions are combined withtherapeutic agents (such as antibiotics) and provided as an aerosol todeliver locally-active drugs to the respiratory system to treat arespiratory disease. In one embodiment, the lower airways are contacted(or contacted exclusively) with the composition. In other embodiments,the composition is used to systemically treat illnesses. Forsystemically-active drugs, the aerosol particles are sized to reach thealveolar surface in peripheral areas of the lung.

In some embodiments, the use of DMSO and/or MSM compositions comprisinga therapeutic agent (such as an antibiotic) is particularly advantageousbecause it provides rapid onset of action. In one embodiment, inhalationdelivery provides a large absorption area of the lung. For locallyacting drugs, the onset of action is immediate in some embodiments.Systemically-active inhaled formulations, according to some embodiments,reach the blood stream quickly. Inhalation therapy provides atherapeutic effect within about 1-90 minutes in some embodiments. In oneembodiment, DMSO and/or MSM enhance the bioavailability of thetherapeutic agent. In a further embodiment, DMSO and/or MSM reduce thedegradation of the therapeutic agent. In another embodiment, aerosolformulations disclosed herein reduce the gastrointestinal side effectsor skin irritation that may occur with oral or topical treatment.

In several embodiments, inhalant particles are sized to minimize thedeposit of those particles by inertial impact into the upper airwayswithout reaching the site of action. In several embodiments, theparticles are sized to minimize deposit in the mouth and throat, therebyminimizing swallowing and undesired local or systemic side effects. Inseveral embodiments, the particles are smaller than 2, 5 or 10 μm. Inone embodiment, the particles are about 3-5 μm and are transported intothe bifurcations and smaller airways of the bronchii and bronchioles. Inanother embodiment, the particles are less than 3 μm and follow theairflow into the alveoli. In several embodiments, the use of DMSO and/orMSM allows for optimizing the particle size of the therapeutic agent.Thus, diseases such as an infectious disease can be more effectivelytreated. Moreover, in several embodiments, the use of DMSO and/or MSMsensitizes drug-resistant microorganisms to antibiotics.

In several embodiments, DMSO and/or MSM form a solution, mixture,emulsion, suspension, or other suitable combination with the therapeuticagent. In one embodiment, homogenization, sonication, high shear fluidprocessing, or other mechanical methods are used to combine thetherapeutic agent with the DMSO and/or MSM. In other embodiments, thetherapeutic agent dissolves readily in DMSO. Unlike other strongsolvents, DMSO is not harmful to lung tissue. Thus, DMSO is especiallyadvantageous in some embodiments because it can both dissolve thetherapeutic agent and deliver said agent without damaging lung tissue.In some embodiments, DMSO dissolves at least 50%, 75%, 90%, 95%, or 99%of the therapeutic agent, and in one embodiment, is able to preventundesired precipitation of the therapeutic agent.

In some embodiments, sprays, gels or wipes comprising DMSO, alone or incombination with MSM, and antibacterial agents are provided forsanitizing medical equipment, surfaces and the body to minimize thespread of infectious disease.

In several embodiments, a pharmaceutical composition comprising DMSOand/or MSM and antimicrobial agents are used as a treatment for aninfectious disease.

In certain embodiments, compositions disclosed herein are effective totreat various infectious diseases including, but not limited to,acinetobacter infection, actinomycosis, Adenovirus infection, Africansleeping sickness (African trypanosomiasis), AIDS, amebiasis,anaplasmosis, Anthrax, Arcanobacterium haemolyticum infection, Argentinehemorrhagic fever, ascariasis, aspergillosis, astrovirus infection,babesiosis, Bacillus cereus infection, bacterial pneumonia, bacterialvaginosis (BV), Bacteroides infection, balantidiasis, Baylisascarisinfection, BK virus infection, black piedra, Blastocystis hominisinfection, blastomycosis, Bolivian hemorrhagic fever, Borreliainfection, botulism, Brazilian hemorrhagic fever, brucellosis,Burkholderia infection, Calicivirus infection, campylobacteriosis,candidiasis (moniliasis; thrush), cat-scratch disease, cellulitis,Chagas disease, chancroid, chickenpox, chlamydia, Chlamydophilapneumoniae infection, cholera, chromoblastomycosis, clonorchiasis,clostridium difficile infection, coccidioidomycosis, Colorado tickfever, common cold, creutzfeldt-Jacob disease, Crimean-Congo hemorrhagicfever, cryptococcosis, cryptosporidiosis, cutaneous larva migrans (CLM),cyclosporiasis, cysticercosis, cytomegalovirus infection, dengue fever,dientamoebiasis, diphtheria, diphyllobothriasis, dracunculiasis, ebolahemorrhagic fever, echinococcosis, ehrlichiosis, enterobiasis (Pinworminfection), Enterococcus infection, enterovirus infection, epidemictyphus, erythema infectiosum, exanthem subitum, fasciolopsiasis,fasciolosis, fatal familial insomnia (FFI), filariasis, food poisoning,free-living amebic infection, Fusobacterium infection, gas gangrene(Clostridial myonecrosis), geotrichosis, Gerstmann-Sträussler-Scheinkersyndrome (GSS), giardiasis, glanders, gnathostomiasis, gonorrhea,granuloma inguinale (Donovanosis), Group A streptococcal infection,Group B streptococcal infection, Haemophilus influenzae infection, hand,foot and mouth disease (HFMD), Hantavirus, Helicobacter pyloriinfection, hemolytic-uremic syndrome (HUS), hemorrhagic fever with renalsyndrome (HFRS), Hepatitis A, B, C, D, or E, herpes simplex,histoplasmosis, hookworm infection, human bocavirus infection, Hhmanewingii ehrlichiosis, human granulocytic anaplasmosis (HGA), humanmetapneumovirus infection, human monocytic ehrlichiosis, humanpapillomavirus (HPV) infection, human parainfluenza virus infection, andhymenolepiasis.

In certain embodiments, formulations disclosed herein are also effectivein treating one or more of the following infectious diseases,Epstein-Barr Virus Infectious Mononucleosis (Mono), Influenza (flu),Isosporiasis, Kawasaki disease, Keratitis, Kingella kingae infection,Kuru, Lassa fever, Legionellosis, Leishmaniasis, Leprosy, Leptospirosis,Listeriosis, Lyme disease, Lymphatic filariasis, Lymphocyticchoriomeningitis, Malaria, Marburg hemorrhagic fever (MHF), Measles,Melioidosis (Whitmore's disease), Meningitis, Meningococcal disease,Metagonimiasis, Microsporidiosis Microsporidia, Molluscum contagiosum(MC), Mumps, Murine typhus, Mycoplasma pneumonia, Mycetoma, Myiasis,Neonatal conjunctivitis, Onchocerciasis (River blindness),Paracoccidioidomycosis (South American blastomycosis), Paragonimiasis,Pasteurellosis, Pediculosis capitis (Head lice), Pediculosis corporis(Body lice), Pediculosis pubis (Pubic lice, Crab lice), Pelvicinflammatory disease (PID), Pertussis (Whooping cough), Plague,Pneumococcal infection, Pneumocystis pneumonia (PCP), Pneumonia,Poliomyelitis, Poliovirus, Primary amoebic meningoencephalitis (PAM),Progressive multifocal leukoencephalopathy, Psittacosis, Q fever,Rabies, Rat-bite fever, Respiratory syncytial virus, Rhinosporidiosis,Rhinovirus infection, Rickettsial infection, Rickettsialpox, Rift Valleyfever (RVF), Rocky mountain spotted fever (RMSF), Rotavirus infection,Rubella, Salmonellosis, SARS (Severe Acute Respiratory Syndrome),Scabies, Schistosomiasis, Sepsis, Shigellosis, Shingles (Herpes zoster),Smallpox, Sporotrichosis, Staphylococcal food poisoning, Staphylococcalinfection, Strongyloidiasis, Syphilis, Taeniasis, Tetanus (Lockjaw),Tinea barbae (Barber's itch), Tinea capitis (Ringworm of the Scalp),Tinea corporis (Ringworm of the Body), Tinea cruris (Jock itch), Tineamanuum (Ringworm of the Hand), Tinea nigra, Tinea pedis (Athlete'sfoot), Tinea unguium (Onychomycosis), Tinea versicolor (Pityriasisversicolor), Toxocariasis (Ocular Larva Migrans (OLM)), Toxocariasis(Visceral Larva Migrans (VLM)), Toxoplasmosis, Trichinellosis,Trichomoniasis, Trichuriasis (Whipworm infection), Tularemia, Ureaplasmaurealyticum infection, Venezuelan equine encephalitis, Venezuelanhemorrhagic fever, Viral pneumonia, West Nile Fever, White piedra,Yersiniosis, Yellow fever, and Zygomycosis.

In several embodiments, compositions disclosed herein are particularlyeffective in treating one or more infectious diseases that are resistantto drug therapies. In addition to those infectious diseases listedabove, which may already be or may become drug resistant in the future,certain embodiments are effective in treating, among others, drugresistant: measles, tetanus, malaria, upper and lower respiratoryinfections, hepatitis, typhoid fever,vancomycin/glycopeptide-intermediate Staphylococcus aureus infection,vancomycin-resistant enterococci, methicillin-resistant Staphylococcusaureus (MRSA), and streptococcus pneumoniae.

In some embodiments, treatment of an infectious disease comprises thepretreatment of a patient with DMSO, followed by the administration of apharmaceutical composition comprising DMSO and antimicrobial agents. Inother embodiments, treatment of an infectious disease comprises thepretreatment of a patient with DMSO, followed by the administration of aformulation comprising DMSO, MSM, and antimicrobial agents. In someembodiments, DMSO pretreatment is administered intravenously via a fastdrip IV catheter. In other embodiments, the DMSO is given in a bolus IVinjection. In yet other embodiments, pretreatment with DMSO is notperformed. Pretreatment compositions additionally include MSM, atherapeutic agent or a combination thereof in some embodiments.

In several embodiments, compositions including DMSO and antimicrobialagents, or DMSO, MSM and antimicrobial agents are administered orally,intravenously, intramuscularly, or subcutaneously. However, as the siteof infection of several infectious diseases is the lungs, in someembodiments, formulations are administered by inhalation. In some suchembodiments, the inhalant means comprises a nebulizer. In otherembodiments, an inhaler is used.

In several embodiments, subjects are pretreated with DMSO usingintravenous DMSO by fast drip within, e.g., a ten minute period. In oneembodiment, DMSO will be provided in glass bottles with proprietarynon-reactive tubing. Subjects will then receive antibiotics dissolved inDMSO in 3 mL doses through an inhaler or mouth spray three times a daywith meals. In one embodiment, DMSO pretreatment is provided in therange of about 25 mg to about 75 mg (e.g., 30 mg, 40 mg, 50 mg, 60 mg,70 mg) in 200 mL 5% dextrose and water. In one embodiment, 56 mg DMSO in200 mL 5% dextrose and water is provided. In one embodiment, thefollowing antibiotics are provided: rifampicin, isoniazid, pyrazinamide,and ethambutol. In one embodiment, about 600 mg rifampicin, 300 mgisoniazid, 2.4 g pyrazinamide, and 1.2 g ethambutol are administered perday, through an inhaler/nebulizer or mouth spray delivered in 3 mLdosages, three times daily. In one embodiment, the antibiotics arecombined with DMSO for delivery via inhalation, with or without thepretreatment with DMSO. Pretreatment with MSM is also provided inseveral embodiments. Intravenous pretreatment of DMSO, MDM, or thecombination of the two is provided in some embodiments. In someexamples, pretreatment formulations include therapeutic agents.

In several embodiments, therapeutic effects are obtained within twoweeks of treatment, within two months of treatment, and/or within sixmonths of treatments. Other therapeutic windows are also provided.

In some embodiments, patients pretreated with DMSO show betterimprovement than those treated with inhalant DMSO and antibioticswithout intravenous DMSO pretreatment. In some embodiments, patientstreated with DMSO with inhalant DMSO and antibiotics show betterimprovement than those treated with antibiotics alone. In severalembodiments, the addition of MSM to the formulation enhances thetherapeutic effects or reduces side effects. In one embodiment, MSM isused alone as a pretreatment.

In several embodiments, compositions disclosed herein are used to notonly treat undesired symptoms and illnesses, but can also act as apreventative agent. For example, formulation may be taken on a regularbasis to prevent the onset of illness. In one embodiment, at risksubjects (e.g., family members or subjects who are exposed to patientshaving an infectious disease) are administered lower doses of DMSOand/or MSM and antibiotics to prevent the onset of infection.

IV. Methods of Use of MSM

Disclosed herein are methods of using any of the disclosed MSMcompositions (as described in Section III) to modulate microbialactivity, such as to enhance or inhibit the activity of microorganisms.For example, methods of enhancing microbial activity are disclosed whichinclude methods of enhancing microbial growth, fermentation efficiency,culturing efficiency, microbial survival, or any combination thereof.Methods of inhibiting microbial activity are also disclosed whichinclude methods of inhibiting microbial growth (such as bacterialgrowth) or infection. In some embodiments, MSM selectively enhances theactivity (e.g., growth) of one microorganism (such as a probioticmicroorganism) and inhibits the activity of undesired microbes (such asan undesired bacterial or fungal activity).

A. Methods of Enhancing Microbial Activity

Methods of enhancing microbial activity are disclosed. In oneembodiment, a method for enhancing activity of a microorganism includesproviding microorganisms, a medium capable of supporting growth of themicroorganisms, and MSM in an amount sufficient to enhance the activity(e.g., fermentation efficiency, growth, culturing efficiency, and/ormicrobial survival) of the microorganisms and contacting the MSM withthe medium, thereby enhancing the growth of the microorganisms in themedium. It is contemplated that the MSM can be added to the medium priorto, concurrent with or after the medium is contacted with themicroorganisms. In one particular embodiment, MSM is provided at aconcentration of about 0.04% to about 5% by weight of the medium or byweight of the moisture content of the medium. As such, in some examples,MSM (such as a composition including about 0.5% to about 5% MSM) is usedto enhance microbial growth. For example, MSM is used to enhancefermentation efficiency, such as to enhance fermentation efficiencyassociated with the production of beer, cider, wine, a biofuel, dairyproduct or any combination thereof. In several examples, MSM enhancesthe production of certain food or beverage making processes that rely onmicroorganisms, such as beer brewing, winemaking, baking, pickling,dairy product production, and the like. In additional examples, MSM isused to enhance the growth of one or more probiotic microorganisms or amicroorganism in a diagnostic test sample. In even further examples, MSMis used to enhance the culturing efficiency and/or survival ofmicroorganisms.

i. Methods to Enhance Microorganism Fermentation Efficiency with MSM

In several embodiments, MSM is used to facilitate energy production.Thus, disclosed herein are methods of enhancing energy production,including methods of enhancing microorganism fermentation efficiency.For example, microorganisms may be used in a fermentation process toproduce ethanol, and in biogas reactors to produce methane. Fermentationis an energy yielding process whereby organic or synthetic molecules aredegraded through metabolism by microorganisms. Some forms ofmicroorganisms, such as bacteria or yeast may be used to convert variousforms of agricultural and urban waste into usable fuels. Microorganismsmay be used as living microbial fuel cells. In some embodiments, MSMenhances bacterial growth and metabolism. In some embodiments, MSMenhances bacterial energy production. In some embodiments, MSM enhancesyeast growth and metabolism. In some embodiments, MSM enhances yeastenergy production.

In several embodiments, MSM is used to activate or enhance one or moreof the following: (i) ethanol fermentation or other anaerobicrespiration used primarily by yeast when oxygen is not present insufficient quantity for normal cellular respiration; (ii) fermentativehydrogen production; (iii) industrial fermentation or other breakdownand re-assembly of biochemicals for industry; (iv) the conversion ofcarbohydrates into alcohols or acids under anaerobic conditions used forfood preparation (e.g., breads, dairy, beans, vinegar, sauerkraut,kimchee, fish, and tofu); (v) fermentation for making brandy, whiskey,vodka, beer, wine or cider, (vi) fermentation for making glucosamine;and (vii) fermentation for the aerobic treatment of tea leaves to breakdown undesired chemicals and develop others that impact, e.g., theflavor and/or nutrients of tea.

In one embodiment, a method of enhancing fermentation efficiency of amicroorganism includes contacting medium containing a microorganismcapable of fermentation with MSM, wherein the MSM is provided at aconcentration of about 0.04% to about 5% by weight of the medium or at aconcentration of about 0.04% to about 5% by weight of the moisturecontent of the medium, wherein the concentration of MSM increases thefermentation efficiency of the microorganism as compared to thefermentation efficiency in the absence of MSM.

In one embodiment, enhanced fermentation efficiency is indicated by anat least 10%, such as about a 20% to 80% increase, about a 30% to 50%increase, including about a 10%, about a 20%, about a 30%, about a 40%,about a 50%, about a 60%, about a 70%, about a 80%, about a 90%, about a100%, about a 150%, about a 200%, about a 300% increase in alcohol,carbon dioxide or acid production in the presence of MSM by themicroorganism as compared to alcohol, carbon dioxide or acid productionin the absence of MSM. For example, the method of enhancing fermentationefficiency is for the production of beer, cider, wine, a biofuel, bread,dairy product or any combination thereof. In some examples, enhancingfermentation efficiency includes an at least 10%, such as about a 20% to80% increase, about a 30% to 50% increase, including about a 10%, abouta 20%, about a 30%, about a 40%, about a 50%, about a 60%, about a 70%,about a 80%, about a 90%, about a 100%, about a 150%, about a 200%,about a 300% increase in production of ethanol, methanol or acombination of thereof as compared to production of ethanol, methanol ora combination of thereof in the absence of MSM. In one particularexample, the microorganism is yeast and the method of enhancingfermentation is for the production of beer. In another example, themicroorganism is algae and the method of enhancing fermentation is forthe production of biofuel.

In some embodiments, enhancing fermentation efficiency includes an atleast 10%, such as about a 20% to 80% increase, about a 30% to 50%increase, including about a 10%, about a 20%, about a 30%, about a 40%,about a 50%, about a 60%, about a 70%, about a 80%, about a 90%, about a100%, about a 150%, about a 200%, about a 300% increase in carbondioxide production in the presence of MSM by the microorganism ascompared to carbon dioxide production in the absence of MSM. In aparticular example, the microorganism is yeast and the method ofenhancing fermentation is for the production of bread.

In additional embodiments, MSM is used to control the fermentationprocess in the production of cultured dairy products such as yogurt,milk, cheese and the like. For example, methods of enhancingfermentation efficiency include an at least 10%, such as about a 20% to80% increase, about a 30% to 50% increase, including about a 10%, abouta 20%, about a 30%, about a 40%, about a 50%, about a 60%, about a 70%,about a 80%, about a 90%, about a 100%, about a 150%, about a 200%,about a 300% increase in lactic acid production in the presence of MSMby the microorganism as compared to lactic acid production in theabsence of MSM.

In some embodiments, the concentration of MSM effective for enhancingfermentation efficiency is about 0.04% to about 5%, such as about 0.1%to about 4%, 0.5% to about 3%, about 1% to about 2%, including about0.04%, to about 0.05%, about 0.06%, about 0.07%, about 0.08%, about0.09%, about 0.1%, about 0.3%, about 0.5%, about 0.7%, about 1%, about1.5%, about 2.0%, about 2.5%, about 3.0%, about 4%, or about 4.5% byweight of medium or by moisture content of the medium. In someembodiments, MSM is added to yeast packages to generate rapid orquick-activating yeast for home or commercial use.

In some examples, the medium for the method of enhancing efficiency of amicroorganism includes a sodium chloride concentration of less than 5%of total moisture content of the medium, such as about 1% to about 3%sodium chloride, including 0%, 0.1%, 0.3%, 0.5%, 0.75%, 1%, 2%, 2.5%, 3%or 4%.

In one certain embodiment, MSM is used for making beer. Yeast culturesare involved in the production of beer during the fermentation processto produce ethanol and carbon dioxide. In some examples, MSM is used toquicken or facilitate activation of the yeast culture, enhancefermentation, reduce potential environmental contamination (such as fromundesirable airborne microorganism) or a combination thereof. Forexample, an increase in the efficiency to activate the yeast (such as anincrease in the efficiency of the starter process), an increase in theefficiency of the fermentation process or combination thereof isindicated by an at least 10%, such as about a 20% to 80% increase, abouta 30% to 50% increase, including about a 10%, about a 20%, about a 30%,about a 40%, about a 50%, about a 60%, about a 70%, about a 80%, about a90%, about a 100%, about a 150%, about a 200%, about a 300% increase ascompared to a control (such as efficiency of these processes in theabsence of MSM).

In several embodiments, MSM is used for enhancing the activity of algae,including the fermentation process associated with generating biofuelfrom use of algae. In one embodiment, this is particularly beneficialfor algaculture (farming algae) for making vegetable oil, biodiesel,bioethanol, biogasoline, biomethanol, biobutanol and/or other biofuels.In one embodiment, the addition of MSM increases the growth rate ofalgae by about 25%, about 30%, about 40%, about 50%, about 100%, about200%, about 300%, about 400%, about 500% or higher. MSM may beparticular advantageous because, by enhancing the activity of algae(such as algal growth), the production of biofuels may become scalable,economically competitive and/or commercially viable. In one embodiment,MSM enhances the process by which the algae product is harvested andconverted into biodiesel. In other embodiments, MSM enhances the processby which the algae's carbohydrate content is fermented into bioethanoland biobutanol. In some embodiments, MSM enhances the algal process by(i) increasing algae yield, (ii) forming more robust algae colonies,(iii) shortening the time to harvest, (iv) shortening the fermentationtime, (v) enhancing fermentation, and/or otherwise supporting orenhancing the growth, reproduction, proliferation, survival rate,metabolism, vitality, robustness, action, and/or function of the algae.Algae, including but not limited to, Botryococcus braunii, Chlorella,Dunaliella tertiolecta, Gracilaria, Pleurochrysis carterae, andSargassum are enhanced by MSM according to several embodiments.

ii. Methods to Enhance Microbial Growth with MSM

In some embodiments, the addition of MSM is particularly advantageousbecause MSM promotes the growth of certain microorganisms (e.g.,probiotics). In some embodiments, microorganisms grown with a mediacomposition comprising MSM have a higher growth rate curve compared to acomparable composition without MSM. In some embodiments, microorganismsgrown with a composition comprising MSM have an increased overallpopulation density compared to a comparable composition without MSM. Incertain embodiments, MSM significantly enhances the simultaneous growthof one or more microorganisms. In some embodiments, media supplementedwith a composition of MSM for enhancing microbial activity (such as aconcentration range of about 0.4% to about 5% or any of the compositionsof MSM for enhancing microbial growth provided in Section III) enhancesthe growth of microorganisms.

Some microorganisms are anaerobic organisms (anaerobes). Anaerobes donot require oxygen for growth. Anaerobes may be used for fermentationand/or culturing. In some embodiments, MSM has a positive impact onanaerobes, such as Bifidobacterium, among others. In some suchembodiments, MSM has a greater positive impact on growth of anaerobesthan other microorganisms. In other embodiments, MSM has a greaterpositive impact on growth of aerobic bacteria as compared to othermicroorganisms. In still other embodiments, aerobes and anaerobes areboth positively impacted by the presence of MSM.

Bacteria can be generally classified as gram-positive or gram-negative,depending on the structure of their cell wall. Gram negative bacteriainclude, but are not limited to, Escherichia coli, Pseudomonas,Salmonella, Shigella, Enterobacteriaceae, Pseudomonas, Moraxella,Helicobacter, Stenotrophomonas, Bdellovibrio, acetic acid bacteria,Legionella, alpha-proteobacteria, cyanobacteria, spirochaetes, greensulfur and green non-sulfur bacteria. Enterics are rod-shapedGram-negative bacteria; most occur normally or pathogenically in theintestines of humans and other animals. In some embodiments, MSM has apositive impact on the growth of gram positive bacteria. In otherembodiments, MSM has a positive impact on the growth of gram negativebacteria. In some such embodiments, MSM has a greater positive impact ongram negative bacteria than gram positive bacteria. In otherembodiments, MSM has a greater positive impact on gram positive bacteriathan gram negative bacteria. In yet other embodiments, MSM has apositive impact on both gram negative bacteria and gram positivebacteria.

Probiotics include live microorganisms thought to be healthy for thehost organism. Lactic acid bacteria (LAB) and bifidobacteria are commontypes of microbes used as probiotics. Certain yeasts and bacilli arealso used. In several embodiments, MSM is used to enhance the survivalor growth of at least one probiotic. Effect on survival of probioticorganisms may be measured on three points according to some embodiments:survivability, colonization, and lactic-acid production. To be effectivein maintaining the health of the gastrointestinal tract, probioticbacteria must be able to survive. Bacterial that are dead on arrival, inmost cases, provide no benefit. Thus in some embodiments, MSM positivelyaffects probiotic survival. In certain embodiments, MSM improves initialsurvival upon exposure of bacteria to a new environment. Thus, in suchembodiments, a product comprising a probiotic and MSM establishes alarger or more healthy (or both) population of probiotic bacteria in thegut as compared to probiotic products alone. In certain embodiments, MSMimproves long-term survival of probiotics. Thus, in such embodiments, aproduct comprising a probiotic and MSM establishes a longer lasting, andbased on growth, a larger population of probiotic bacteria in the gut ascompared to probiotic products alone. Of those probiotic bacteriaarriving in the gut alive, those that colonize (multiply in) the gutgenerally provide benefit. Thus, in several embodiments, MSM improvesthe speed and frequency of probiotic multiplication. In still otherembodiments, MSM increases the production of lactic acid.

In some embodiments, MSM has a positive impact on probiotic growth. Insome embodiments, MSM has a positive impact on the microbial flora ofthe gastrointestinal tract. In some such embodiments, MSM has a positiveimpact on intestinal health. In some embodiments, foods containingprobiotics are supplemented with MSM, and the resulting probiotic levelsachieved in the intestinal tract are greater than after ingestion of theprobiotic-containing food alone. In some such embodiments, the additionof MSM results in higher level of probiotic organism in a shorter timeframe with ingestion of probiotic-containing food alone. In someembodiments, probiotics that require 24 to 48 hours before effects areobserved are rendered more efficacious because MSM increases theirlifespan.

Bacterial growth typically has an initial lag phase where the bacteriaadjust to the environment, before going into the log phase, where cellsdouble. After the log phase there is a stationary phase. During thestationary phase, the growth rate slows as a result of nutrientdepletion and accumulation of metabolic by-products. This phase isreached as the microbes begin to exhaust the resources that areavailable to them. This phase is a relatively constant value as the rateof microbial growth is equal to the rate of microbial death. In thedeath phase, bacteria typically exhaust nutrients and population numbersdrop.

In some embodiments, MSM impacts the lag phase, log phase, stationaryphase, death phase or any combination thereof. In certain embodiments,MSM shortens the lag phase, so that the bacteria, such as probioticbacteria, begin the log phase at an earlier time. In severalembodiments, MSM extends the stationary phase. In certain embodiments,the death rate is slowed in the presence of MSM. Certain embodiments ofthe disclosure as described herein positively affect one or more, and incertain embodiments all, phases of the growth of probiotic bacteria.

In some embodiments, MSM impacts metabolism of microbes (e.g.,probiotics), in the lag phase. During the lag phase, microbes arematuring (growing in size) and not yet able to divide (thus not growingin number). During the lag phase of the microbial growth cycle,synthesis of RNA, enzymes and other molecules occurs. In someembodiments, MSM decreases the duration of the lag phase by acceleratingthe maturation (and adaptation of microorganisms to environmentalstressors) of the microorganisms, thereby allowing microbial divisionsooner than in MSM-free media.

In some embodiments, MSM-supplementation results in an increase in thelog phase of growth of microbes (e.g., probiotics). The exponentialphase (sometimes called the log phase) of growth is a periodcharacterized by cell doubling. The number of new microbes appearing perunit time is proportional to the present population. If growth is notlimited, doubling will continue at a constant rate so both the number ofcells and the rate of population increase doubles with each consecutivetime period. Exponential growth cannot continue indefinitely, however,because the medium is soon depleted of nutrients and enriched withwastes. In some embodiments, MSM increases the overall duration of theexponential phase. In other embodiments, the presence of MSM in thegrowth media promotes microbial entry into the exponential phase morequickly than microorganisms in MSM-free media. The initial growthenvironment with MSM-supplemented media can be conducive to cellmultiplication and survival.

In several embodiments, MSM affects the stationary phase of microbial(e.g., probiotic) growth. In one example, MSM-supplementation of mediaextends the stationary phase for microbes as compared to MSM-free media.

In some embodiments, MSM enhances probiotic growth, which in turn crowdsout and takes nutrients from undesired microbes. In other embodiments,MSM enhances probiotic activity, which in turn enhances lactic andacetic acid production to lower the environmental pH and inhibit theactivity of undesirable bacteria. In further embodiments, MSM enhancesprobiotic activity, which in turn stimulates the production ofimmunomodulating agents (e.g., cytokines), thereby enhancing the immuneresponse. In certain embodiments, MSM enhances probiotic activity, whichin turn enhances bactericidal activity with respect to undesiredmicrobial contamination. In one embodiment, MSM enhances probioticgrowth at a faster rate than undesired microbes, thereby allowingprobiotics to preferentially colonize an environment (e.g., edibleproducts, intestinal tract).

Without being bound by a particular theory, in several embodiments, MSMhas a biochemical effect on microbial metabolism. For example, in someembodiments, the addition of MSM has a positive effect on metabolism ofcertain microorganisms such that certain microorganisms are better ableto adapt and/or recover from environmental changes. In some embodiments,MSM serves as a substrate or cofactor for microbial metabolism and/oranapleurotic biochemical pathways. In some embodiments, MSM positivelyimpacts the lag phase of growth. In some embodiments, MSM increases thelog phase of microbial growth. In still further embodiments, MSMincreases the duration of the stationary phase of microbial growth. Insome embodiments, MSM decreases the rate of population decline ofcertain microbes. In certain embodiments, MSM provides a selective orsemi-selective growth environment, so that certain microbial speciesgrow more rapidly (or to attain larger population size, or both) ascompared to other microbial species. In certain embodiments, MSM impactsthe metabolic activity of microorganisms, while in other embodiments,MSM creates an environment more conducive to microbial growth.

As such, methods of enhancing microbial growth are provided. In someembodiments, methods of enhancing microbial growth include in vitromethods for enhancing the growth of one or more microorganisms. In oneexample, in vitro methods for enhancing growth of one or moremicroorganisms includes contacting one or more microorganisms with amedium capable of supporting growth of the one or more microorganisms;and providing MSM to the medium at about 0.4% to about 5% by weight ofthe medium or by weight of moisture content of the medium therebyenhancing the growth of the one or more microorganisms in vitro ascompared to growth of the one or more microorganisms in vitro in theabsence of MSM. It is contemplated that similar methods can be used forenhancing the growth of desired microorganisms (such as probiotics) invivo. For example, an increase in microbial growth is indicated by anincrease in weight of the microorganism or cell number such as an atleast 10%, such as about a 20% to 80% increase, about a 30% to 50%increase, including about a 10%, about a 20%, about a 30%, about a 40%,about a 50%, about a 60%, about a 70%, about a 80%, about a 90%, about a100%, about a 150%, about a 200%, about a 300% increase as compared to acontrol (such as weight of the microorganism or cell number in theabsence of MSM). Increases in microorganism growth can be detected bymethods known to those of skill in the art including those described inthe Examples.

a. Methods for Enhancing Growth of a Probiotic Microorganism

Methods for enhancing growth of one or more probiotic microorganisms aredisclosed. For example, methods of enhancing growth of one or moreprobiotic microorganisms include contacting one or more probioticmicroorganisms with a medium capable of supporting growth of one or moreprobiotic microorganisms; and providing MSM to the medium at about 0.4%to about 5% by weight of the medium or by weight of moisture content ofthe medium thereby enhancing the growth of the one or moremicroorganisms as compared to growth of the one or more microorganismsin the absence of MSM. In one example, the concentration of MSM is about1% to about 3% of the weight of the medium or by weight of the moisturecontent of the medium. An increase in probiotic growth is indicated byan at least 10%, such as about a 20% to 80% increase, about a 30% to 50%increase, including about a 10%, about a 20%, about a 30%, about a 40%,about a 50%, about a 60%, about a 70%, about a 80%, about a 90%, about a100%, about a 150%, about a 200%, about a 300% increase in cell growthas compared to a control (such as cell growth in the absence of MSM)

In some examples, the medium for enhancing microbial growth, such asprobiotic growth, include a probiotic-containing product, such as milk,yogurt, rice yogurt, frozen yogurt, chocolate, cheese, beer, wine,vinegar, sauerkraut or any combination thereof.

It is contemplated that the method can be used to enhance growth of anyprobiotic microorganism, including, but not limited to Lactobacillusacidophilus, Lactobacillus delbrueckii, Bacillus coagulans,Lactobacillus rhamnosus, Bifidobacteruim bifidum or any combinationthereof. In one embodiment, the disclosed methods are used to enhancethe activity of the bacteria Lactobacillus rhamnosus. In otherembodiments, the disclosed methods are used to enhance the activity ofspecies within the Lactobacillus genus. For example, a method ofenhancing the activity (e.g., growth) of Lactobacillus acidophilusincludes contacting Lactobacillus acidophilus with a medium capable ofsupporting growth of Lactobacillus acidophilus; and providing MSM to themedium at about less than about 1% (such as at about 0.04%, 0.05%. 0.1%,0.2%, 0.3%, 0.4%, 0.5%. 0.6%, 0.75, 0.8% or 0.9%) by weight of themedium or by weight of a moisture content of the medium therebyenhancing the growth of Lactobacillus acidophilus as compared to growthof Lactobacillus acidophilus in the absence of MSM.

In other embodiments, the disclosed methods are used to enhance theactivity of Bifidobacterium bifidum. For example, a method of enhancingthe activity (e.g., growth) of Bifidobacterium bifidum includescontacting Bifidobacterium bifidum with a medium capable of supportinggrowth of Bifidobacterium bifidum; and providing MSM to the medium atabout less than about 1% (such as at about 0.04%, 0.05%. 0.1%, 0.2%,0.3%, 0.4%, 0.5%. 0.6%, 0.75, 0.8% or 0.9%) of weight of the medium or amoisture content of the medium thereby enhancing the growth ofBifidobacterium bifidum as compared to growth of Bifidobacterium bifidumin the absence of MSM.

An increase in probiotic growth is indicated by an increase in weight ofthe probiotic microorganism or cell number of such, including an atleast 10%, such as about a 20% to 80% increase, about a 30% to 50%increase, including about a 10%, about a 20%, about a 30%, about a 40%,about a 50%, about a 60%, about a 70%, about a 80%, about a 90%, about a100%, about a 150%, about a 200%, about a 300% increase as compared to acontrol (such as weight of the probiotic microorganism or cell number inthe absence of MSM). Increases in probiotic microorganism growth can bedetected by methods known to those of skill in the art including thosedescribed in the Examples.

b. Methods for Enhancing Growth of a Microorganism in a Diagnostic TestSample or Industrial Test Sample

Methods for enhancing growth of a microorganism in a diagnostic testsample or industrial test sample are disclosed. In one embodiment, amethod for enhancing growth of a microorganism in a diagnostic testsample is provided. In one example, the method includes contacting adiagnostic test sample (e.g., blood, tissue, scrapings, bodily fluidsand metabolic products, and the like) comprising one or moremicroorganisms with a medium capable of supporting growth of the one ormore microorganisms; and providing MSM to the medium at a concentrationsufficient to enhance microbial growth, thereby enhancing the growth ofthe one or more microorganisms in the diagnostic test sample as comparedto growth of the one or more microorganisms in the absence of MSM.

In some embodiments, a method for enhancing growth of a microorganism inan industrial test sample is provided. In one example, the methodincludes contacting an industrial test sample (e.g., water sample,household mold or bacteria sample and other like samples) comprising oneor more microorganisms with a medium capable of supporting growth of theone or more microorganisms; and providing MSM to the medium at aconcentration sufficient to enhance microbial growth, thereby enhancingthe growth of the one or more microorganisms in the industrial testsample as compared to growth of the one or more microorganisms in theabsence of MSM.

In several embodiments, MSM is provided in a composition forfacilitating diagnostic assays or industrial test sample assays, such asat a concentration of about 0.04% to about 5% by weight of the sample orby weight of the moisture content of the sample. In some embodiments,MSM is provided in a composition for facilitating diagnostic assays orindustrial test sample assays such as any of the compositions of MSMcapable of enhancing microbial activity which are described in SectionIII. In certain embodiments, MSM is added directly to the diagnostic orindustrial test sample comprising microorganisms.

According to several embodiments described herein, MSM can shortendetection and/or analysis time by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%or 90%. According to several embodiments described herein, MSM canenhance microbial activity (such as growth) by at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 100-fold, 500-foldor 1000-fold. For example, an increase in microbial growth is indicatedby an increase in weight of the microorganism or cell number of such,including an at least 10%, such as about a 20% to 80% increase, about a30% to 50% increase, including about a 10%, about a 20%, about a 30%,about a 40%, about a 50%, about a 60%, about a 70%, about a 80%, about a90%, about a 100%, about a 150%, about a 200%, about a 300% increase ascompared to a control (such as weight of the microorganism or cellnumber in the absence of MSM). Increases in microorganism growth can bedetected by methods known to those of skill in the art including thosedescribed in the Examples.

In several embodiments, MSM is used in conjunction with medicalscreening tests and rapid diagnostic testing, such as in urine or bloodsamples. In many cases, diagnostic tests are performed to identifypossible microbial infections. Several groups of microorganisms,including bacteria, viruses, mold, and yeast, can cause infections. If amicroorganism is found, more testing is done to determine whichantibiotics may be effective in treating the infection. In orderdiagnose such infections as early as possible, in some embodiments MSMis used to supplement the growth media used in diagnostic tests toincrease the rate of growth of microorganisms in the patient sample,thereby improving the detection time of the test. In some embodiments,MSM may enhance detection sensitivity of a diagnostic test. In someembodiments, the diagnostic test is a urine test. In some embodiments,the diagnostic test is a blood test. In other embodiments, other patientsamples can be grown for diagnostic purposes, such as sputum, saliva,skin scrapings, dental swaps, vaginal or cervical swabs, and the like.In one embodiment, MSM is used to provide a rapid strep test. Forexample, a sample of bodily fluid (the diagnostic test sample) is addedto a test tube or culture dish (the medium). The medium supports theculture of any microbes that may exist in the bodily fluid. By providinga medium that is pre-dosed with MSM or by adding MSM prior to or afteradding the bodily fluid to the test tube or culture dish, microbes inthe bodily fluid (or their assayable products or metabolites) wouldincrease, and would be easier to assay. Thus, diagnosis is facilitated.

In several embodiments, the use of MSM facilitates medical diagnosis ofviral infections by supporting the growth of viruses for diagnosticassay. Viruses include, but are not limited to, human immunodeficiencyvirus, herpes simplex virus, papilloma virus, parainfluenza virus,influenza, hepatitis, and other viruses. Likewise, medical diagnosis ofother infections, such as those caused by bacteria, fungi, yeast andparasites are also facilitated by MSM according to several embodiments.The use of MSM facilitates vaccine development in one embodiment.

In several embodiments, MSM is used to enhance detection of microbes ina commercial or industrial test. Microorganisms are a common watercontaminant. Many water safety test kits evaluate quality of drinkingwater through Environmental Protection Agency (EPA) testing methods bytesting for, among others, the presence of bacteria. Mold found in thehome, office and school environments has been linked to pulmonarydisorders, and allergic symptoms. However, some tests used to detectbacteria or mold can be time consuming for analysis while some testsadditionally detect only viable (living) organisms. Thus, in severalembodiments MSM is used to supplement growth media used in commercialdetection tests. In some embodiments, MSM-supplemented media improvesthe detection time of tests. In some embodiments, MSM-supplemented mediaimproves the detection sensitivity of such tests. In certainembodiments, MSM restores environmentally stressed bacteria which werepreviously non-viable. In still further embodiments, diagnostic testkits comprising microorganism-specific MSM-supplemented media are usedto enhance the detection time or sensitivity of a test directed todetecting a particular microorganism. In other embodiments, MSM is usedto supplement a broad spectrum growth media, such that a variety ofmicroorganisms are detected more rapidly or with increased sensitivity.

iii. Methods for Enhancing Survivability of Microorganisms and Cellswith MSM

Methods for enhancing survivability of microorganisms (including, butnot limited to probiotic microorganisms) or cells (such as, stem cellsor recombinant cells) are disclosed. For example, methods of enhancingsurvivability of microorganisms or cells, such as cells in culture,include contacting one or more microorganisms or selected cells with MSMat about 0.4% to about 5% by weight of the medium or by weight of amoisture content of the medium thereby enhancing the survivability ofthe one or more microorganisms or collection of cells as compared tosurvivability of the one or more microorganisms or collection of cellsin the absence of MSM. In one example, the concentration of MSM is about1% to about 3% of the weight of the medium or the moisture content ofthe medium. An increase in survivability is indicated by an at least10%, such as about a 20% to 80% increase, about a 30% to 50% increase,including about a 10%, about a 20%, about a 30%, about a 40%, about a50%, about a 60%, about a 70%, about a 80%, about a 90%, about a 100%,about a 150%, about a 200%, about a 300% increase in colony or cellnumber as compared to a control (such as colony or cell number in theabsence of MSM).

According to several embodiments, MSM improves initial survival of themicroorganisms (including, but not limited to, probioticmicroorganisms). In one embodiment, MSM improves long-term survival ofthe microorganisms. In one embodiment, MSM extends the stationary phaseof a growth curve of the microorganisms.

In several embodiments, MSM extends the shelf-life of a product byextending the lifespan of beneficial bacteria as compared to productswithout MSM. For example, a probiotic containing product may have ashelf-life of several weeks, after which time the probiotic organismsbegin to decline in health and/or population. However, in someembodiments, the addition of MSM to a probiotic containing productincreases the length of time from product packaging until the decline ofprobiotic health and/or population. In such embodiments, the probioticproduct is functional (in terms of delivering a population of health andactive probiotics to the GI tract of the consumer) for a longer periodof time after packaging.

In several embodiments, the addition of MSM increases the time tospoilage of ingestible products by supporting or enhancing the activityof beneficial microbes, with a resulting decrease in the activity ofundesired microbes. For example, MSM can increase the shelf life ofedible products, such as a probiotic product, by about 10% to 100%(e.g., 20%, 30%, 40%, 50%, 75%, 150%, 200% or more). For example, in oneembodiment, if the shelf life of an edible product is 10 days, theaddition of MSM will increase the shelf life to at least 11 days in someembodiments (e.g., 11 days, 14 days, 15 days, 20 days, or 25 days). As afurther example, in another embodiment, if an edible product has a shelflife of 14 days at room temperature and/or 30 days in the refrigeratorand/or 3 months in the freezer, the addition of MSM will increase theshelf life to 30 days at room temperature and/or 60 days in therefrigerator and/or 6 months in the freezer. In some embodiments, theuse of MSM unexpectedly enhances the activity of beneficial microbes andinhibits (either directly or indirectly) the activity of undesiredbacteria, thereby reducing or eliminating the need for sterilization(e.g., by irradiation, filtration, heat, chemicals, etc.).

In some embodiments, MSM is provided to enhance the activity of geneticvectors, such as recombinant viral vectors in recombinant cells. Thismay be beneficial for diagnostics as well as therapeutics, such as genetherapy. In some embodiments, MSM is used to enhance the activity (e.g.,growth, culture, or viability) of one or more plasmid vectors, binaryvectors, cloning vectors, expression vectors, shuttle vector, and viralvectors. As such, methods for enhancing gene therapy are disclosed inwhich one more processes associated with gene therapy is enhanced orincreased by treating the recombinant cell or microorganism with aconcentration of MSM (such as a concentration of about 0.04% to about 5%MSM) capable of enhancing one or more processes of gene therapy (such asthe expression, growth or survivability of recombinant cells ormicroorganisms), thereby increasing the effectiveness of the genetherapy.

iv. Methods of Enhancing Culturing Efficiency with MSM

Methods of enhancing culturing efficiency with MSM are disclosed herein.In one embodiment, methods for enhancing various types of cultures areprovided, including, but not limited to enhancing antibiotic, steroid,cell (e.g., recombinant and wild-type), microorganism and fertilizerculturing efficiency. For example, in several embodiments, MSM is usedto supplement culture media used for the growth or propagation ofmicrobial organisms. In several embodiments, MSM-supplemented mediaenhances the culture efficiency by enhancing the growth of the cells.

In some embodiments, methods of enhancing culturing efficiency includeenhancing/promoting microbial activity in environmental and industrialfields. Microorganisms participate in element cycles such as the carboncycle and nitrogen cycle, as well as fulfilling other vital roles invirtually all ecosystems, such as recycling the waste products and/orremains of other organisms through decomposition. Thus, in someembodiments, the use of MSM may enhance waste decomposition and wastemanagement. Many biological oxidation processes for treating industrialwastewaters have in common the use of oxygen (or air) and microbialaction. Specially-cultured microbes are used in the biological treatmentof sewage and industrial waste effluent, a process known asbioaugmentation. Bioaugmentation is used to ensure that the in situmicroorganisms can degrade contaminants. In some embodiments, MSMenhances certain microorganisms' degradation of contaminants. In someembodiments, MSM is added to gardening products, such as soil,fertilizers, and compost bins, to enhance the activity of beneficialmicroorganisms. As such, MSM is used to increase the efficiency offertilizers and compost reactions.

In one embodiment, a method for enhancing the efficiency of a fertilizerincludes applying MSM to medium in an amount sufficient to enhance theactivity of a fertilizer, thereby enhancing the activity of thefertilizer. In one particular embodiment, MSM is dissolved in a solutionto a final concentration of about 0.04% to about 5%. This solution isthen sprayed onto a plant surface either prior to, following orsimultaneously as the fertilizer. An increase in fertilizer efficiencyis indicated by an at least 10%, such as about a 20% to 80% increase,about a 30% to 50% increase, including about a 10%, about a 20%, about a30%, about a 40%, about a 50%, about a 60%, about a 70%, about a 80%,about a 90%, about a 100%, about a 150%, about a 200%, about a 300%increase in plant growth as compared to a control (such as plant growthin the absence of MSM).

In another embodiment, a method for enhancing the efficiency ofcomposting is disclosed. This method includes applying MSM to thecompost in amount sufficient to enhance the activity of one or moremicroorganisms or substances present in the compost. In one particularembodiment, MSM is dissolved in a solution to a final concentration ofabout 0.04% to about 5%. This solution is then applied to the compost(such as by pouring or spraying the solution) and allowed timesufficient to enhance the efficiency of the composting. An increase incompost efficiency is indicated by an at least 10%, such as about a 20%to 80% increase, about a 30% to 50% increase, including about a 10%,about a 20%, about a 30%, about a 40%, about a 50%, about a 60%, about a70%, about a 80%, about a 90%, about a 100%, about a 150%, about a 200%,about a 300% increase in nitrate levels as compared to a control (suchas nitrate levels in the absence of MSM). In other examples, an increasein compost efficiency is indicated by an at least 10%, such as about a20% to 80% increase, about a 30% to 50% increase, including about a 10%,about a 20%, about a 30%, about a 40%, about a 50%, about a 60%, about a70%, about a 80%, about a 90%, about a 100%, about a 150%, about a 200%,about a 300% decrease in the amount of time that decomposition oforganic matter occurs as compared to a control (such as decompositionrate in the absence of MSM).

B. Methods of Inhibiting Microbial Activity

Methods of inhibiting microbial activity are disclosed. In oneembodiment, a method for inhibiting microbial activity includesselecting a medium that is susceptible to contamination; and contactingthe medium with MSM at a concentration of about 6% to about 16% ofweight by volume, thereby inhibiting microbial activity as compared tomicrobial activity in a control (such as microbial activity in theabsence of MSM). By an at least 10%, such as about a 20% to 80%decrease, about a 30% to 50% decrease, including about a 10%, about a20%, about a 30%, about a 40%, about a 50%, about a 60%, about a 70%,about a 80%, about a 90%, about a 100%, about a 150%, about a 200%,about a 300% decrease as compared to a control (such as microbialactivity in the absence of MSM).

In some embodiments, a method for inhibiting microbial activity includesselecting a medium that is susceptible to bacterial contamination; andcontacting the medium with MSM at a concentration of about 6% to about16% of weight by volume, thereby inhibiting bacterial activity. In someembodiments, a method for inhibiting microbial activity includesselecting a medium that is susceptible to viral contamination (such ascontamination by human immunodeficiency virus, H1N1, herpes simplexvirus, papilloma virus, parainfluenza virus, influenza, hepatitis, orother like viruses); and contacting the medium with MSM at aconcentration of about 6% to about 16% of weight by volume, therebyinhibiting viral activity.

In one particular example, a method of inhibiting microbial activityincludes selecting a medium that is susceptible to H1N1 influenzacontamination; and contacting the medium with MSM at a concentration ofabout 10% to about 16% of weight by volume, thereby inhibiting H1N1influenza microbial activity. In some embodiments, MSM inhibits themicrobial activity by reducing growth rate of H1N1 influenza by at least10%, such as by about a 20%, about a 30%, about a 40%, about a 50%,about a 60%, about a 70%, about a 80%, about a 90%, about a 100%, abouta 150%, about a 200%, about a 300% decrease in H1N1 influenza growth orinfectivity as compared to a control (such as H1N1 influenza activity orinfectivity in the absence of MSM).

In several embodiments, the methods include MSM at about 8% (by weight)or greater, of a product's total weight or moisture content. In certainembodiments, MSM is an effective antimicrobial agent when used atconcentrations between about 5% and about 16%. In certain embodiments,MSM is an effective antimicrobial agent when used at concentrations(based on a product's total weight or moisture content) between about 9%and about 16%, between about 10% and about 16%, between about 12% andabout 16%, between about 9% and about 13%, and between about 10% andabout 12%. In certain embodiments MSM is an effective antimicrobialagent when used at concentrations between about 5% and about 16%,including 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14, and 15%. In severalembodiments disclosed herein, percentages of MSM are based on aproduct's moisture content. In some embodiments, MSM is particularlyeffective when combined with water or other liquid components. Inseveral embodiments, the percentages of MSM provided herein are based onthe amount of a polar solvent in a product or other medium.

In some embodiments, the disclosed methods of inhibiting microbialactivity include inhibition of growth of specific microorganisms. Insome examples, the methods include inhibiting growth of a wide range ofmicroorganisms in certain media or products. In some embodiments,log-scale reductions are realized after the first 24 hours. In someother embodiments, significant log-scale reductions are evident within24-48 hours. In some embodiments, the disclosed methods include MSMformulations which yield reduction in microbial (e.g., bacterial) levelsranging from about 0.5 log to about 5 log or more within two weeks. Insome embodiments, the disclosed methods of inhibiting microbial activityresult in a log reduction between about 1 log reduction and about 3 logor more reductions. In other embodiments, the disclosed methods ofinhibiting microbial activity lethally inhibit the growth of certainmicroorganisms. In one embodiment, a method using a formulation of MSMbetween about 12% and about 16% lethally kills certain microbes (e.g.,bacteria) within about 48 hours. In another embodiment, a formulationcomprising MSM between about 8% and about 12% lethally kills certainmicrobes (e.g., bacteria) within about three to seven days. In otherembodiments, the methods employ a formulation of MSM between about 5%and about 8%, combined with a reduced amount of conventionalpreservative, which lethally kills certain microbes (e.g., bacteria)within about 48 hours. With higher preservative concentrations, MSMlevels may be reduced further.

In some embodiments, the disclosed methods of inhibiting microbialactivity with MSM (such as with about 6% to about 16% MSM) impactmetabolism of microbes in the lag phase. For example, the disclosedmethod increases the duration of the lag phase. An alteration, such asan increase in the lag phase, may be detected by methods of those knownto skill in the art including those described in the Examples.

In some embodiments, MSM-supplementation results in a decrease in thelog phase of growth of microbes. The exponential phase (sometimes calledthe log phase) of growth is a period characterized by cell doubling. Thenumber of new microbes appearing per unit time is proportional to thepresent population. If growth is not limited, doubling will continue ata constant rate so both the number of cells and the rate of populationincrease doubles with each consecutive time period. Exponential growthcannot continue indefinitely, however, because the medium is soondepleted of nutrients and enriched with wastes. In some embodiments, MSMdecreases the overall duration of the exponential phase. In otherembodiments, the presence of MSM in the growth media inhibits microbialentry into the exponential phase.

In several embodiments, the disclosed methods of inhibiting microbialactivity include modulating the stationary phase of microbial growth.During stationary phase, the growth rate slows as a result of nutrientdepletion and accumulation of metabolic by-products. This phase isreached as the microbes begin to exhaust the resources that areavailable to them. This phase is a relatively constant value as the rateof microbial growth is equal to the rate of microbial death.MSM-supplementation of media at certain concentrations shortens thestationary phase for microbes in one embodiment.

It is contemplated that a medium includes any medium or environmentcontaining or suitable for supporting contamination including, but notlimited to, cosmetics, broths, agar, cultures, foods, beverages, cellsuspensions, biological tissue, biological fluids, inorganic surfaces,organic surfaces, substrates, living cells, host cells, diagnosticassays, and other solid, liquid, matrix, gelatinous, or gaseousenvironments. In some examples, the medium is a bodily fluid, a bodilytissue, or a surface.

In some embodiments, contacting the medium include topical, oral,intravenous, intramuscular, or subcutaneous administration of MSM to themedium susceptible to microbial contamination. In other embodiments,contacting the medium includes spraying or wiping the medium susceptibleto microbial contamination with a disclosed MSM composition/formulation.For example, a surface can include any surface susceptible tocontamination including, but not limited to, a household surface, anindustrial surface (such as surfaces in public restrooms, door handles,floors, walls, hand railings, shopping carts and the like), bedding,coverings, industrial equipment or surface, blood, skin or a combinationthereof. For example, a household surface may include a door handle,door knob, a trash can, a counter top, floor, toilet seat or any surfacewhich is commonly touched or exposed to possible contaminates.

Unintended microbial growth may occur in many cosmetics, health andbeauty aids, topical products, and oral products. Acute or continued useof products with microbial contamination can lead to adverse healtheffects for the user. Contamination may occur, for example, duringmanufacturing, packaging, or repetitive use by a consumer which includesrepeated opening and closing of containers, contact with hands, skin, ormucous membranes, or repeated withdrawal/administration of individualdoses. In the absence of antimicrobial properties, these products mayallow the unintended growth of many different, and potentiallydeleterious, microorganisms.

Antimicrobial preservatives may be added to products to protect themfrom microbial growth. Common general use antimicrobial preservativesinclude calcium propionate, sodium nitrate, sodium nitrite, sulfites(sulfur dioxide, sodium bisulfite, potassium hydrogen sulfite, etc.) anddisodium EDTA. Cosmetic preservatives include formaldehyde, potassiumsorbate, methylparaben, and methylchloroisothiazolinone.

In many cases, preservatives must be added in a minimum effectiveconcentration, as adverse reactions may occur at certain concentrationsor doses. Thus, while preservatives may inhibit microbial growth, theyalso have the potential to cause chemical burns and/or irritate the skinand mucous membranes. Some modern synthetic preservatives have becomecontroversial because they have been shown to cause respiratory or otherhealth problems. Addition of certain preservatives to commercialproducts may present unique complications with solubility, pH limits,de-activation by some polyethylene glycol (PEG) compounds, and a shiftin the color, consistency or fragrance of a product. Some preservativeshave only limited activity against particular classes of microorganisms.

Methods of inhibiting microbial activity in a consumer product are alsodisclosed. In one embodiment, the method includes selecting a mediumthat is susceptible to microbial contamination, such as a consumerproduct, and adding MSM to the medium to affect the microbialcontamination by inhibiting microbial activity. MSM is provided in aconcentration of at least 10% according to one embodiment (e.g., 10-16%,16-20%, 20-30%, 30-40%, 40-50%, 50-75% or higher, and overlapping rangesthereof). The medium is free from preservatives in some embodiments.

In some embodiments, methods of inhibiting microbial activity in acosmetic cream at room temperature are provided. In one embodiment, themethod includes selecting a medium that is susceptible to microbialcontamination; and adding MSM to the medium to affect the microbialcontamination by inhibiting microbial activity. MSM is added in aconcentration of at least 5% according to one embodiment (e.g., 5-10%,10-16%, 16-20%, 20-30%, 30-40%, 40-50%, 50-75% or higher, andoverlapping ranges thereof). The medium is free from preservatives insome embodiments. The medium includes a cosmetic cream in someembodiments. In one example, MSM inhibits microbial activity by at least50% in the cosmetic cream at room temperature.

In some examples, the medium includes one or more of the following:cosmetics, broths, agar, cultures, foods, beverages, cell suspensions,biological tissue, biological fluids, inorganic surfaces, organicsurfaces, substrates, living cells, host cells, diagnostic assays, andother solid, liquid, matrix, gelatinous, or gaseous environments. Forexample, in one embodiment, the medium includes an optical product or aproduct for oral hygiene or health. The medium may also include a bodilyfluid or tissue, such as blood. In one example, the medium is sterilizedbefore adding MSM and/or after adding MSM. In other examples, nosterilization is needed. In some examples, the antimicrobial propertiesof MSM reduce or eliminate the need for sterilization.

In some examples, microbial contamination is caused by bacteria, such asgram positive bacteria and/or gram negative bacteria, fungi, parasites,yeast, mold, viruses, or combinations thereof (e.g., bacteria and mold,or other combinations). In several embodiments, microbial contaminationis caused by one or more of the following genera: Candida, Aspergillus,Escherichia, Pseudomonas, Staphylococcus, and Streptococcus, orcombinations thereof. In other embodiment, microbial contamination iscaused by an infectious disease including any of the infectious diseasesdescribed herein.

In several embodiments, methods for treating an infectious disease aredisclosed including, but not limited to H1N1, herpes simplex virus, orHIV. In one embodiment, the method includes administering a therapeuticeffective amount of a therapeutic agent and DMSO alone, MSM alone or acombination of DMSO and MSM. The concentration of DMSO and/or MSM rangesfrom about 6% to about 17% in a composition.

In some embodiments, MSM inhibits microbial activity by reducing thegrowth rate of one or more microbes by more than 50% which in turnincreases the shelf life of the medium. It is contemplated that MSM canconfer a therapeutic and/or aesthetic benefit. In some embodiments, thetherapeutic or aesthetic benefit is unrelated to the microbialinhibition.

In some embodiments, the disclosed methods of inhibition of microbialactivity inhibit microbial activity at temperatures conducive tomicrobial activity, including 20-25° C., 25-30° C., 30-40° C., 40-50° C.and higher (and overlapping ranges thereof). In some embodiments, MSMinhibits microbial activity at humidity levels favorable for microbialactivity, including 50%-60%, 60-70%, 70-80%, 80-95%, and higher (andoverlapping ranges thereof).

MSM is particularly advantageous in several embodiments because it maybe used in higher concentrations than other preservatives, which whenused even in low concentrations can cause adverse effects. For example,preservatives have been implicated in atopic dermatitis, rashes,flushing, abdominal pain, nausea, asthma, rhinitis, muscle aches, jointaches, fatigue, numbness, migraines, attention deficit and hyperactivitydisorder, palpitations and arrhythmias. By contrast, MSM is not known tocause such effects in concentrations provided according to preferredembodiments herein. Moreover, MSM has a dual function according to someembodiments. Not only does MSM inhibit the growth of undesiredmicroorganisms, MSM also beneficially affects the product to which it isadded in several embodiments.

In some embodiments, the disclosed methods of inhibiting microbialactivity not only inhibit microbial activity, but provide one or morebeneficial effects, including, but not limited to, reduction of musclecramps, skin irritation, reduction of pain, joint lubrication, reductionof inflammation, rheumatoid arthritis and osteoarthritis treatment,cardiovascular improvements, skin lubrication, improved wound healing,and improved scalp, hair, cuticle, and nails.

In some embodiments, the disclosed methods of inhibiting microbialactivity are used to prevent or minimize the formation of new microbes.In other embodiments, the methods are used to kill or reduce existingmicrobes. In one embodiment, MSM can convert an otherwise unusablecontaminated product into a usable product.

According to several embodiments, the methods inhibit microbial activityinstantaneously. In other embodiments, the methods inhibits microbialactivity at and up to 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 10days, 14 days, 1 month, 3 months, 6 months, 1 year, 2 years, 3 years, 4years, 5 years and longer.

In several embodiments, MSM is added to cleaning agents to enhanceantimicrobial activity (e.g., to inhibit microorganism activity). Insome embodiments, MSM is added to a soap formulation. In someembodiments, the product is a dry soap while in other embodiments, theproduct is a liquid soap. In some embodiments, MSM is added to a gelformulation to yield a sanitizer. For example, methods of inhibitingmicrobial activity include methods of sanitizing a surface, such as thebody, equipment, floors, materials, walls, etc. In certain embodiments,the resultant sanitizer is an instant sanitizer. In other embodiments,the sanitizer acts non-instantaneously (e.g., is effective over time).In some embodiments, the sanitizer is applied to the body. In yet otherembodiments, the product is applied to a surface. Surfaces include, butare not limited to, commercial surfaces, medical devices, medicalsurfaces, production equipment, production floors and food preparationsurfaces. Surfaces may include, but not limited to household surfaces,vehicles, computers, clothing, and toys.

In some further embodiments, methods of inhibiting microbial activityinclude spraying or incorporating MSM (e.g., about 5% to about 50% intoface masks or filters. Filters may include, but are not limited to,air-conditioner filters, air filters, water filters. Environments withrecycled air, such as airplanes, may especially benefit from MSMfiltration systems. Waste treatment and water filtration plants may alsoincorporate MSM to inhibit microbial activity. In some embodiments, MSMis provided to reduce microbial contamination in flower arrangements andgardening products (such as fertilizers and soils).

In some embodiments, methods of inhibiting microbial activity includeinhibiting microbial activity of a microorganism present on animal feedand to prevent microbial growth during storage or processing of thefeed. Types of animal feed include, but are not limited to, compoundfeed, fodder, or forage. Animal feed may consist of raw materials and/oradditives. Raw feed may be provided as hay or grains. Alternatively, rawmaterial may be manufactured and provided as meal, pellets or crumbles.In some embodiments, MSM is applied to animal feed to reduce moldgrowth. In other embodiments, MSM is applied to animal feed to reducefungal growth. In some embodiments, MSM is applied to raw feed materialsand thus incorporated into a finished feed product. In furtherembodiments, the product is applied to feed during or aftermanufacturing. In some embodiments, the product is applied to feed forlong-term storage.

V. Methods of Making Products Including MSM

Methods of making products including MSM are disclosed herein. In someembodiments, MSM is incorporated at a stage that will reducecrystallization of MSM. In one embodiment, MSM is incorporated into aproduct prior to emulsification of said product. In another embodiment,MSM is encapsulated (e.g., in a lipid, polymer, or other material) priorto addition to a product. Microencapsulated MSM, according to someembodiments, may be designed to time or dose-release MSM. In yet otherembodiments, MSM is combined with the aqueous portion of a product priorto mixing of the wet and dry ingredients. In one embodiment, MSM in drypowder form is mixed in a matrix with an aqueous or polar liquid toactivate the MSM.

In yet another embodiment, MSM is added to a product at an elevatedtemperature (e.g., greater than 25° C., 30° C., 40° C., 50° C., 75° C.,or higher). In some embodiments, MSM is materially unaffected by heat,and can be added prior to heating. Solutions that have a temperature ofgreater than about 35° C. support MSM concentrations greater than 50% insome embodiments. In several embodiments, MSM does not substantiallyimpact the pH of the product to which it is added. In one embodiment,hygroscopic solid products and other products with low moisture contentcomprise MSM in the range of about 15% or higher.

Methods for manufacturing a product having a reduced preservativeconcentration are also disclosed herein. In one embodiment, the methodincludes providing a medium that is susceptible to microbialcontamination, wherein the medium comprises a preservative and addingMSM to the medium, wherein the MSM affects the microbial contaminationby inhibiting microbial growth. MSM is added in a concentration of atleast 5% to about 20% according to one embodiment (e.g., 5-8%, 8-12%,12-15%, 15-20%, or higher, and overlapping ranges thereof). In oneembodiment, MSM and the preservative inhibit microbial growth by atleast 50% in the medium at room temperature, and MSM supplements orenhances the ability of the preservative to inhibit microbial growth,thereby reducing the concentration of preservative needed to inhibitmicrobial growth. In one embodiment, the medium is emulsified orotherwise mixed. In one embodiment, MSM is added to the medium prioremulsification (or other mixing).

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the disclosure to the particular features or embodimentsdescribed.

EXAMPLES Example 1 MSM-Based Modulation of Microbial Activity

This example describes MSM-based modulation of microbial activity, suchas enhancing or inhibiting microbial growth depending upon theconcentration of MSM.

Side-by-side microbial growth studies were conducted in mediasupplemented with MSM at a concentration of 0.1% to 10% and a controlsample containing 0% MSM. The microorganisms evaluated were Aspergillusniger, Candida albicans, Staphylococcus aureus, Pseudomonas aeruginosa,Escherichia coli, and Salmonella cholerasuis. All microorganisms weregrown in tryptic soy broth (TSB) and with the exception of Candida andAspergillus, all were successively transferred into fresh media each dayfor 4 consecutive days prior to inoculation to maintain organisms in anexponential growth phase. Candida and Aspergillus had 48-58 hours ofgrowth in TSB prior to inoculation into the test media. Aspergillus wasalso grown on multiple potato dextrose agar plates (PDA) for 48-58hours. The Aspergillus inoculum was prepared by taking a surface rinsewith TSB off of the PDA plates with a lawn of Aspergillus, and thenadded to the 48-58 hour culture until turbid. For each testmicroorganism, 90 mL aliquots of TSB were prepared with either 10% or 0%MSM. Once each set of test media was plated for sterility, they wereinoculated at a level of 5 μL1 of inoculum per 10 mLs of broth (1:2000inoculum dilution) with each respective microorganism. Bacterialorganisms were incubated at 30° C.±2° C. and fungal organisms wereincubated at 25° C.±2° C.

Fungal organisms were plated daily on PDA at days 0 through day 7 every24 hours. Preparation and plating were conducted at room temperature.Fungal plates were incubated at 25° C.±2° C. for at least 3 days. Testsamples were plated in triplicate at each test date and the averages arereported. Data are expressed as recovered colony forming units permilliliter (cfu/mL).

The effects of MSM on Aspergillus niger growth and Candida albicansgrowth are shown in Tables 1-1(a) and 1-1(b), respectively. Ten percentMSM inhibited Aspergillus niger growth by day 4 of treatment asrepresented by a dramatic reduction in colony formation on such date. Areduction in growth was also observed in Candida albicans MSM treatedsamples; however the reduction was not as dramatic as compared with theAspergillus niger. For example, 10% MSM supplemented media resulted inreduced yeast viability as compared to lower concentrations of MSM asearly as 2 days (plating day 2 for Candida). Candida growth was reducedearly on in the study, with substantial reductions in fungal populationat Day 4. After these time points, the divergence in growth curvescontinued throughout the study for Candida. These data indicate that aconcentration of 10% MSM provides a significant negative effect on thegrowth of various fungal organisms over time.

TABLE 1-1(a) Effect of MSM on Aspergillus niger Growth. Aspergillusniger 0% 0.1% 0.5% 1.0% 10% MSM MSM MSM MSM MSM Day 0 1.0 × 10³ 9.1 ×10² 7.6 × 10² 8.9 × 10² 1.2 × 10³ Day 1 1.3 × 10³ 2.3 × 10³ 2.7 × 10³2.0 × 10³ 7.0 × 10² Day 2 4.3 × 10³ 3.0 × 10² 4.0 × 10³ 2.0 × 10³ 1.6 ×10² Day 3 2.0 × 10³ 7.5 × 10² 1.3 × 10³ 1.0 × 10³ 4.6 × 10² Day 4 1.4 ×10⁴ 5.0 × 10³ 3.7 × 10³ 3.3 × 10³ 20 Day 5 1.3 × 10⁴ 4.3 × 10³ 9.7 × 10³6.0 × 10³ 10 Day 6 4.4 × 10⁴ 1.1 × 10⁴ 7.0 × 10³ 4.7 × 10³ 3 Day 7 4.1 ×10⁴ 1.3 × 10⁴ 7.0 × 10³ 5.7 × 10³ 3

TABLE 1-1(b) Effect of MSM on Candida albicans Growth. Candida albicans0% 0.1% 0.5% 1.0% 10% MSM MSM MSM MSM MSM Day 0 2.2 × 10⁴ 2.1 × 10⁴ 1.8× 10⁴ 2.5 × 10⁴ 2.6 × 10⁴ Day 1 1.0 × 10⁵ 4.7 × 10⁶ 4.9 × 10⁶ 5.0 × 10⁶<1.0 × 10⁵  Day 2 1.0 × 10⁷ 9.8 × 10⁶ 1.1 × 10⁷ 9.8 × 10⁶ 4.0 × 10³ Day3 1.3 × 10⁷ 1.2 × 10⁷ 1.3 × 10⁷ 1.2 × 10⁷ <1.0 × 10⁵  Day 4 1.9 × 10⁷1.3 × 10⁷ 1.4 × 10⁷ 1.2 × 10⁷ 7.0 × 10⁴ Day 5 1.6 × 10⁷ 1.3 × 10⁷ 1.4 ×10⁷ 1.3 × 10⁷ 1.7 × 10⁴ Day 6 8.8 × 10⁶ 1.3 × 10⁷ 1.4 × 10⁷ 1.6 × 10⁷2.8 × 10³ Day 7 1.7 × 10⁷ 1.4 × 10⁷ 1.6 × 10⁷ 1.7 × 10⁷ 3.3 × 10³

The effect of MSM on Staphylococcus aureus growth is illustrated inTable 1-2. A difference in viability in the presence of higherconcentrations of MSM was observed. In particular, 10% MSM appeared toboth slow the growth rate and the maximum population size ofStaphylococcus aureus.

TABLE 1-2 Effect of MSM on Staphylococcus aureus Growth. Staphylococcusaureus 0% 0.1% 0.5% 1.0% 10% MSM MSM MSM MSM MSM Day 0 2.4 × 10⁵ 2.4 ×10⁵ 2.5 × 10⁵ 2.3 × 10⁵ 2.5 × 10⁵ Day 1 3.6 × 10⁸ 4.6 × 10⁸ 4.1 × 10⁸6.0 × 10⁸ 5.3 × 10⁷ Day 2 7.5 × 10⁸ 8.5 × 10⁸ 7.8 × 10⁸ 8.2 × 10⁸ 3.0 ×10⁸ Day 3 9.6 × 10⁸ 9.1 × 10⁸ 8.4 × 10⁸ 9.6 × 10⁸ 5.1 × 10⁸ Day 4 6.4 ×10⁸ 7.9 × 10⁸ 4.7 × 10⁸ 4.5 × 10⁸ 3.7 × 10⁸ Day 7 2.6 × 10⁸ 1.8 × 10⁸1.7 × 10⁸ 3.3 × 10⁸ 9.0 × 10⁷

The effect of MSM on Pseudomonas aeruginosa growth is illustrated inTable 1-3. Ten percent MSM media supplementation resulted in substantialdivergence in Pseudomonas aeruginosa viability over time. For example,10% MSM supplemented media yielded a population reduction that lastedfor the first 4 days of the study, but did not persist after such time.

TABLE 1-3 Effect of 10% MSM on Pseudomonas aeruginosa Growth.Pseudomonas aeruginosa 0% 0.1% 0.5% 1.0% 10% MSM MSM MSM MSM MSM Day 04.0 × 10⁵ 4.8 × 10⁵ 4.8 × 10⁵ 4.2 × 10⁵ 4.0 × 10⁵ Day 1 4.4 × 10⁸ 5.1 ×10⁸ 5.4 × 10⁸ 5.8 × 10⁸ 1.0 × 10⁵ Day 2 1.1 × 10⁹ 6.0 × 10⁸ 9.2 × 10⁸5.7 × 10⁸ 5.8 × 10³ Day 3 1.6 × 10⁹ 1.4 × 10⁹ 1.2 × 10⁹ 1.0 × 10⁹ 5.3 ×10³ Day 4 1.6 × 10⁹ 2.0 × 10⁹ 1.4 × 10⁹ 1.9 × 10⁹ 3.7 × 10⁶ Day 7 1.4 ×10⁹ 2.1 × 10⁹ 1.6 × 10⁹ 1.2 × 10⁹ 6.0 × 10⁶

The effect of MSM on Pseudomonas aeruginosa growth is illustrated inTable 1-4. Ten percent MSM media supplementation resulted insubstantially less growth of E. coli over time.

TABLE 1-4 Effect of 10% MSM on Escherichia coli Growth. Escherichia coli0% 0.1% 0.5% 1.0% 10% MSM MSM MSM MSM MSM Day 0 6.9 × 10⁵ 6.4 × 10⁵ 7.0× 10⁵ 6.7 × 10⁵ 6.8 × 10⁵ Day 1 9.2 × 10⁸ 1.0 × 10⁹ 1.4 × 10⁹ 1.2 × 10⁹3.0 × 10⁴ Day 2 1.3 × 10⁹ 1.4 × 10⁹ 1.6 × 10⁹ 3.2 × 10⁹ 1.2 × 10⁵ Day 31.6 × 10⁹ 2.0 × 10⁹ 1.9 × 10⁹ 1.8 × 10⁹ 3.9 × 10⁷ Day 4 1.4 × 10⁹ 1.4 ×10⁹ 1.4 × 10⁹ 1.4 × 10⁹ 1.2 × 10⁸ Day 7 1.3 × 10⁹ 1.2 × 10⁹ 2.8 × 10⁹1.5 × 10⁹ 7.0 × 10⁷

The effect of MSM on Salmonella cholerasuis growth is illustrated inTable 1-5. Media supplemented with 10% MSM reduced the growth ofSalmonella cholerasuis for the majority of the time points of the study.

TABLE 1-5 Effect of 10% MSM on Salmonella cholerasuis Growth. Salmonellacholerasuis 0% 0.1% 0.5% 1.0% 10% MSM MSM MSM MSM MSM Day 0 6.8 × 10⁵8.7 × 10⁵ 5.9 × 10⁵ 5.5 × 10⁵ 7.6 × 10⁵ Day 1 9.6 × 10⁸ 1.2 × 10⁹ 9.7 ×10⁸ 1.1 × 10⁹ 1.7 × 10⁶ Day 2 1.3 × 10⁹ 1.0 × 10⁹ 1.2 × 10⁹ 1.3 × 10⁹7.7 × 10⁷ Day 3 1.2 × 10⁹ 1.2 × 10⁹ 1.5 × 10⁹ 1.5 × 10⁹ 3.2 × 10⁸ Day 47.8 × 10⁸ 6.0 × 10⁸ 7.1 × 10⁸ 8.0 × 10⁸ 1.9 × 10⁸ Day 7 3.0 × 10⁸ 3.4 ×10⁸ 3.6 × 10⁸ 9.2 × 10⁸ 2.3 × 10⁸

These studies indicate that certain concentrations of MSM inhibitgrowth, including Aspergillus niger, Candida albicans, Staphylococcusaureus, Pseudomonas aeruginosa and E. coli growth.

Example 2 Antimicrobial Effectiveness Testing of MSM-Supplemented Media

This example describes the antimicrobial effectiveness testing resultsof MSM-supplemented media.

Compounds or formulated products having antimicrobial activity may beevaluated with the United States Pharmacopeia (USP) AntimicrobialEffectiveness Test (AET). The AET involves the addition of specifiedmicroorganisms (Candida albicans, Aspergillus niger, Escherichia coli,Pseudomonas aeruginosa and Staphylococcus aureus) directly to a testproduct at relatively high concentrations to simulate contamination. Theproduct is held for one month, with weekly analysis of microorganismlevels. Depending on the route of administration of a product,satisfaction of the AET generally requires a 1 to 3 log reduction inbacteria from initial levels, which should occur in one to two weeks,with no further increase in bacteria after two weeks. For yeast andmold, no increase from the initial inoculums level is permitted.Successfully meeting the criteria of the AET demonstrates that aproduct, optionally supplemented with an antimicrobial compound underevaluation, can withstand an inoculation of up to one millionmicroorganisms per gram of product without becoming contaminated. TheAET demonstrates the effectiveness of a preservative system in a productand/or may be used as part of a stability study to determine if apreservative system will affect a product's shelf life.

The AET was performed by adding the specified microorganisms directly toMSM supplemented test media at concentrations simulating microbialcontamination. Fresh, active cultures standardized to a concentrationbetween 100,000 to 1,000,000 cells per mL of the test product, wereadded to the MSM supplemented media. Inoculations were made usingCandida albicans, Aspergillus niger, Escherichia coli, Pseudomonasaeruginosa and Staphylococcus aureus. Tryptic soy broth (TSB) was usedas the culturing media. MSM was diluted 1/1, 1/5, 1/10, 1/100, and1/1000 and each used to supplement the media. The inoculated media washeld for one month, during which time the added microorganisms wereenumerated weekly to determine if they were growing, dying off, orremaining near the initial inoculation level. Data points were measuredin triplicate at 48 hours, 3, 5, 14, 20, 28, and 30 days. The results ofthese studies are shown in Tables 2-1 through 2-8. The acceptancecriteria for antimicrobial effectiveness are described in detail in theUSP, herein incorporated by reference.

TABLE 2-1 AET test results for 1:1 dilution of MSM. Test Initial 20 2830 Organism Inoculum 48 hrs 3 days 5 days 14 days days days daysAspergillus   3 × 10⁵ 3.5 × 10⁵ 3.2 × 10⁵ 3.1 × 10⁵ 2.3 × 10³ <10 <10<10 Niger Candida 2.5 × 10⁵ 2.8 × 10⁵ 2.6 × 10⁵ 2.4 × 10⁵ 1.2 × 10³ <10<10 <10 Albicans Escherichia 1.3 × 10⁵ 1.3 × 10⁵ 1.2 × 10⁵ 1.1 × 10⁵ 1.8× 10³ <10 <10 <10 coli Pseudomonas 1.5 × 10⁵ 1.8 × 10⁵ 1.9 × 10⁵ 1.6 ×10⁵ 4.0 × 10³ <10 <10 <10 aeruginosa Staphylococcus 5.5 × 10⁵ 5.4 × 10⁵5.6 × 10⁵ 5.2 × 10⁵ 3.7 × 10³ <10 <10 <10 aureus Salmonella 4.6 × 10⁵4.9 × 10⁵ 5.1 × 10⁵ 4.7 × 10⁵ 2.5 × 10³ <10 <10 <10 typhimurium

TABLE 2-2 Log Reduction from Initial Microorganism Inoculum with 1:1dilution of MSM. Test Organism 14 Days 28 Days Aspergillus Niger 3.4 4.5Candida Albicans 3.0 4.4 Escherichia coli 3.3 4.1 Pseudomonas aeruginosa3.6 4.2 Staphylococcus aureus 3.6 4.7 Salmonella typhimurium 3.4 4.7

TABLE 2-3 AET test results for 1:5 dilution of MSM. Test Initial 20 2830 Organism Inoculum 48 hrs 3 days 5 days 14 days days days daysAspergillus   3 × 10⁵ 4.0 × 10⁵ 4.2 × 10⁵ 3.8 × 10⁵   3 × 10³ <10 <10<10 Niger Candida 2.5 × 10⁵ 2.9 × 10⁵ 3.1 × 10⁵ 3.2 × 10⁵   2 × 10³ <10<10 <10 Albicans Escherichia 1.3 × 10⁵ 1.4 × 10⁵ 1.7 × 10⁵   2 × 10⁵   1× 10³ <10 <10 <10 coli Pseudomonas 1.5 × 10⁵ 1.6 × 10⁵ 2.0 × 10⁵ 2.2 ×10⁵ 1.2 × 10³ <10 <10 <10 aeruginosa Staphylococcus 5.5 × 10⁵ 5.7 × 10⁵6.0 × 10⁵ 5.9 × 10⁵ 2.3 × 10³ <10 <10 <10 aureus Salmonella 4.6 × 10⁵5.0 × 10⁵ 5.2 × 10⁵ 5.4 × 10⁵ 3.2 × 10³ <10 <10 <10 typhimurium

TABLE 2-4 Log Reduction from Initial Microorganism Inoculum with 1:5dilution of MSM. Test Organism 14 Days 28 Days Aspergillus Niger 3.5 4.5Candida Albicans 3.3 4.4 Escherichia coli 3.0 4.1 Pseudomonas aeruginosa3.2 4.2 Staphylococcus aureus 3.4 4.7 Salmonella typhimurium 3.5 4.7

TABLE 2-5 AET test results for 1:10 dilution of MSM. Initial 20 28 30Test Organism Inoculum 48 hrs 3 days 5 days 14 days days days daysAspergillus   3 × 10⁵   5 × 10⁵ 5.2 × 10⁵ 5.4 × 10⁵   2 × 10⁴ <10 <10<10 Niger Candida 2.5 × 10⁵   3 × 10⁵ 3.4 × 10⁵   4 × 10⁵ 1.5 × 10⁴ <10<10 <10 Albicans Escherichia 1.3 × 10⁵   2 × 10⁵ 2.3 × 10⁵ 3.2 × 10⁵ 2.5× 10³ <10 <10 <10 coli Pseudomonas 1.5 × 10⁵ 1.9 × 10⁵ 2.2 × 10⁵ 2.9 ×10⁵   4 × 10⁵ <10 <10 <10 aeruginosa Staphylococcus 5.5 × 10⁵ 5.9 × 10⁵6.1 × 10⁵ 6.3 × 10⁵ 2.9 × 10⁴ <10 <10 <10 aureus Salmonella 4.6 × 10⁵5.0 × 10⁵ 5.2 × 10⁵ 5.5 × 10⁵ 3.2 × 10³ <10 <10 <10 typhimurium

TABLE 2-6 Log Reduction from Initial Microorganism Inoculum with 1:10dilution of MSM. Test Organism 14 Days 28 Days Aspergillus Niger 4.3 4.5Candida Albicans 4.2 4.4 Escherichia coli 3.4 4.1 Pseudomonas aeruginosa3.6 4.2 Staphylococcus aureus 4.5 4.7 Salmonella typhimurium 3.5 4.7

TABLE 2-7 AET test results for 1:100 dilution of MSM. Test OrganismInitial Inoculum 48 hrs Aspergillus Niger  3 × 10⁵ TNTC* CandidaAlbicans 2.5 × 10⁵ TNTC Escherichia coli 1.3 × 10⁵ TNTC Pseudomonasaeruginosa 1.5 × 10⁵ TNTC Staphylococcus aureus 5.5 × 10⁵ TNTCSalmonella typhimurium 4.6 × 10⁵ TNTC *Colonies too numerous to count(TNTC)

TABLE 2-8 AET test results for 1:1000 dilution of MSM. Test OrganismInitial Inoculum 48 hrs Aspergillus Niger  3 × 10⁵ TNTC* CandidaAlbicans 2.5 × 10⁵ TNTC Escherichia coli 1.3 × 10⁵ TNTC Pseudomonasaeruginosa 1.5 × 10⁵ TNTC Staphylococcus aureus 5.5 × 10⁵ TNTCSalmonella typhimurium 4.6 × 10⁵ TNTC

The 1:1, 1:5, and 1:10 dilutions of MSM (Tables 2-1 to 2-6, above),indicate that these concentrations of MSM in the media killedmicroorganisms and do not simply have a static effect on growth. Basedon the culture populations at day 5, bactericidal effects wereunexpected, as the culture populations were stable or showed signs ofincreasing growth. However, by 14 days, reduction in initial inoculationlevels were observed, and by 20 days, a total kill of all microorganismswas observed using at least 10% MSM. These results were confirmed byspiking a 90 mL TSB blank with 10 mLs of the tested dilution matrix(e.g., the media believed to no longer contain any livingmicroorganisms). None of the microorganisms could be cultured andcontamination was not observed. These results demonstrate that MSM, atcertain concentrations, is bactericidal to these organisms.

Example 3 Bactericidal Effects of Sterile and Non-Sterile MSM onEscherichia coli

This example describes the bactericidal effects of sterile andnon-sterile MSM on E. coli growth.

The USP <51> AET test methodology as described in Example 2 was used asthe basis to evaluate the lethality to Escherichia coli (ATCC strain8739) of various concentrations of MSM ranging from 5 to 16% in TSB orsaline. The USP <51> AET is a compendial (United States Pharmacopeia)antimicrobial effectiveness test method for determining if apreservative is effective based upon a verified and validatedmethodology. The parameters of the AET are described above. In thisstudy, after the designated incubation period, cultures were evaluatedvisually and were then streaked and grown on selective MacConkey agarfor qualitative analysis of the effects of the various MSMconcentrations. This study also evaluated the effect of pre-sterilizingthe MSM (by steam autoclaving at 121° C. for 15 minutes) prior to mediapreparation. Test media were prepared by weighing an appropriate amountof MSM and adding it to 25 mL of TSB media or saline. Media compositionswere coded as provided in Table 3-1.

TABLE 3-1 Media compositions tested against E. coli. Media CodePreparation NS Sterile saline added to non-sterile MSM NSA Saline + MSM,then sterilized TSB Sterile TSB added to non-sterile MSM TSBA TSB + MSM,then sterilized TSBC TSB without MSM, spiked with E. coli (−) TSBCnegative control (no E. coli) NSC Saline without MSM, spiked with E.coli (−) NSC negative control (no E. coli)

All tubes except the negative controls were spiked with 250 μl of a1.2×10⁸ culture, which yields an initial E. coli population density of1.2×10⁶/mL. Tubes were incubated at 25° C.

At 24 hours, visible signs of growth were observed in the 5-9% MSM inTSB/TSBA media (see Table 3-2 below). In contrast, no signs of growthwere seen in the tubes with 10-16% MSM in TSB/TSBA media (see Table 3-2below). Saline tubes did not show any signs of growth. When streaked onMacConkey media, heavy growth resulted in all media containing 5-9% MSM,while fewer colonies were detected on the streaked plates from the 10%MSM in TSB/TSBA media. Little to no growth resulted from the streakingof the 11-16% MSM in TSB/TSBA media. Growth was detected in the TSB andsaline positive control (TSB or saline without MSM, spiked with E.coli), while no growth was detected on the plates streaked from thenon-spiked media.

TABLE 3-2 Growth profile of E. coli in MSM- containing media after 24hours. MSM Concentration TSB TSBA NS NSA  5% Heavy Heavy Heavy Heavy  6%Heavy Heavy Heavy Heavy  7% Heavy Heavy Heavy Heavy  8% Heavy HeavyHeavy Heavy  9% Heavy Heavy Heavy Heavy 10% Moderate Moderate HeavyHeavy 11% Few Few Heavy Heavy 12% Few Few Moderate Moderate 13% Few FewModerate Moderate 14% Few Few Moderate Moderate 15% Few Few ModerateModerate 16% Few Few Moderate Moderate

As shown in Table 3-3, at 48 hours signs of heavy growth were observedin the 5-10% MSM in TSB/TSBA culture tubes. Apparent growth was observedin the 11% MSM in TSB/TSBA culture tubes. Similar to the 24 hour timepoint, little to no observable signs of growth were observed in the12-15% MSM in TSB/TSBA culture tubes. After streaking, heavy bacterialgrowth occurred in all media containing 5-10% MSM. 11% MSM in TSB/TSBAallowed moderate growth, while the same concentration of MSM added tosaline allowed heavy growth. At concentrations from 12-16% MSM inTSB/TSBA, little to no growth was detected on the plates. Moderategrowth was observed from similar concentrations of MSM in saline media.

TABLE 3-3 Growth profile of E. coli in MSM- containing media after 48hours. MSM Concentration TSB TSBA NS NSA  5% Heavy Heavy Heavy Heavy  6%Heavy Heavy Heavy Heavy  7% Heavy Heavy Heavy Heavy  8% Heavy HeavyHeavy Heavy  9% Heavy Heavy Heavy Heavy 10% Heavy Heavy Heavy Heavy 11%Moderate Moderate Heavy Heavy 12% Few Few Moderate Moderate 13% Few FewModerate Moderate 14% Few Few Moderate Moderate 15% Few Few ModerateModerate 16% Few Few Moderate Moderate

After 72 hours of culturing, signs of heavy growth were observed in the5-10% MSM in TSB/TSBA culture tubes (Table 3-4). Apparent growth wasobserved in the 11% MSM in TSB/TSBA culture tubes. Similar to the 24hour time point, little to no observable signs of growth were observedin the 12-15% MSM in TSB/TSBA culture tubes. After streaking, heavybacterial growth occurred in all media containing 5-10% MSM. 11% MSM inTSB/TSBA allowed moderate growth, while the same concentration of MSMadded to saline allowed heavy growth. At concentrations from 12-16% MSMin TSB/TSBA, little to no growth was detected on the plates. Moderategrowth was observed from similar concentrations of MSM in saline media.

TABLE 3-4 Growth profile of E. coli in MSM- containing media after 72hours. MSM Concentration TSB TSBA NS NSA  5% Heavy Heavy Heavy Heavy  6%Heavy Heavy Heavy Heavy  7% Heavy Heavy Heavy Heavy  8% Heavy HeavyHeavy Heavy  9% Heavy Heavy Heavy Heavy 10% Heavy Heavy Heavy Heavy 11%Moderate Moderate Heavy Heavy 12% Few Few Moderate Moderate 13% Few FewModerate Moderate 14% Few Few Moderate Moderate 15% Few Few ModerateModerate 16% Few Few Moderate Moderate

These results show that concentrations of MSM from about 10-16% areeffective at killing bacteria at certain time points. At 24 hours, 10%MSM reduced the viable bacterial population, while at 48-72 hours higherconcentrations were more effective at killing the majority of thebacterial population. The 10-16% concentrations of MSM in TSB/TSBA weremore effective than the same concentration in a saline based media. Thedata further suggest that steam sterilization does not inherently impactthe effectiveness of MSM.

Example 4 Comparison of MSM Bactericidal Effectiveness in Saline orTryptic Soy Broth(TSB)-Based Media

This example compares MSM bactericidal effectiveness in saline andTSB-based media.

As presented in Examples 2 and 3, the USP <51> AET test methodology wasused as the basis to evaluate the lethality to E. coli of variousconcentrations of MSM (flaked or microprill) ranging from 5 to 16% inTSB or saline. Each media composition was inoculated with 1.25×10⁶/mL ofE. coli and then cultured for seven days at 35° C. At the end of theincubation period, cultures were evaluated visually and were then grownon trypto-soy agar at serially diluted concentrations to ensurebacterial growth (if any) at a density that was able to be quantified.Plated cultures were grown for 24 hours at 35° C. before analysis. Mediacompositions were coded as shown in Table 4-1 and results of thesestudies are provided in Table 4-2.

TABLE 4-1 Media compositions Media Code Preparation PS Microprill MSMadded to saline FS Flaked MSM added to saline PTSB Microprill MSM addedto TSB FTSB Flaked MSM added to TSB TSBC TSB without MSM, spiked with E.coli (−) TSBC negative control (no E. coli) NSC Saline without MSM,spiked with E. coli (−) NSC negative control (no E. coli)

TABLE 4-2 Log growth of E. coli in different media with variousconcentrations of MSM. MSM Concentration FTSB FS PTSB PS 16% 1 4.5 1 4.815% 2.6 4.8 2.3 5.1 14% 2.9 4.9 2.1 5.6 13% 2.6 5.5 3 5.6 12% 2.7 5.1 35.8 11% 3.6 5.4 3.3 5.9 10% 6 5.5 6 5.8  9% 6 5.6 6 5.9  8% 6 5.7 6 6 7% 6 5.9 6 6  6% 6 5.9 6 6.2  5% 6 5.7 6 6.4

Concentrations in the range from 11-16% MSM have a negative effect onthe growth of E. coli in cultured for 7 days. As the concentration ofMSM increased above 10% in either the FTSB or PTSB media, E. coli growthwas reduced. Both forms of MSM showed efficacy in inhibiting bacterialgrowth.

Example 5 Effect of Sodium Chloride-Free Media on Bactericidal Effect ofMSM

This example shows the effect of sodium chloride-free media onbactericidal effects of MSM.

A study employing Müller-Hinton broth media, which does not containNaCl, was performed. Standard Müller-Hinton media was compared toMüller-Hinton media supplemented with NaCl to the same level as thesaline-based media of Example 4. MSM was added to each media inconcentrations ranging from 5-16%. After inoculation of eachMSM-containing media type with 1.9×10⁷ cfu/mL of E. coli, the cultureswere incubated at 35° C. for seven days. Aliquots of each culture weretaken at 24 and 48 hours, as well as 7 days. Aliquots were grown ontrypto-soy agar at serially diluted concentrations to ensure bacterialgrowth (if any) at a density that was able to be quantified. Platedcultures were grown for 24 hours at 35° C. before analysis. Mediacompositions were coded as provided in Table 5-1. Results of thesestudies are provided in Tables 5-2 through 5-4.

TABLE 5-1 Media compositions Media Code Preparation PMHS Microprill MSMadded to Müller-Hinton media plus NaCl FMHS Flaked MSM added toMüller-Hinton media plus NaCl PMH Microprill MSM added to Müller-Hintonmedia FMH Flaked MSM added to Müller-Hinton media MHC Müller-Hintonmedia without MSM, spiked with E. coli (−) MHC negative control (no E.coli) MHNSC Müller-Hinton media plus NaCl without MSM, spiked with E.coli (−) MHNSC negative control (no E. coli)

After 24 hours in culture, an approximately 1 log reduction from theinitial inoculum was detected in all media having MSM concentrationsgreat than 13% (Table 5-2). Further, at 12% MSM, all media compositionsreduced the E. coli growth, except the PMHS composition. At 11% MSM,only the FMH media reduced E. coli growth.

TABLE 5-2 Log growth of E. coli in MSM supplemented Müller-Hinton mediaor Müller-Hinton (plus NaCl) after 24 hours MSM Concentration PMHS FMHSPMH FMH 16% 6.2 6.1 6.1 6.2 15% 6.0 6.1 6.0 6.2 14% 6.0 6.0 5.9 6.3 13%6.3 6.2 5.8 5.9 12% 7.2 5.7 5.8 5.9 11% 7.9 7.8 7.4 5.9 10% 8.7 8.2 7.97.8  9% 8.3 8.3 8.3 8.2  8% 8.2 8.4 8.4 8.4  7% 8.3 8.4 8.4 8.5  6% 8.18.4 8.5 8.5  5% 8.2 8.4 8.6 8.6

After 48 hours in culture, media compositions with MSM concentrationsgreater than 13% reduced E. coli growth by 1-2 logs. Certainconcentrations of MSM are effective at reducing bacterial growth, whichis surprising because other concentrations of MSM are effective atsupporting increased bacterial activity.

TABLE 5-3 Log growth of E. coli in MSM supplemented Müller-Hinton mediaor Müller-Hinton (plus NaCl) after 48 hours MSM Concentration PMHS FMHSPMH FMH 16% 6.0 5.9 6.0 5.9 15% 6.0 5.9 5.8 4.6 14% 5.7 5.9 5.4 5.3 13%5.9 5.9 5.4 5.2 12% 6.7 6.9 5.6 5.3 11% 7.8 7.8 7.2 7.1 10% 7.9 8.0 7.98.2  9% 8.2 8.1 8.1 8.2  8% 8.1 8.1 8.2 8.3  7% 8.2 8.2 8.0 8.4  6% 8.18.2 8.4 8.4  5% 8.4 8.3 8.3 8.4

After 7 days in culture, media compositions containing as low as 12% MSMsubstantially inhibited the growth of E. coli (Table 5-4). FMHS mediawas most efficacious at 12% MSM, yielding a 3 log reduction in E. coli.

TABLE 5-4 Log growth of E. coli in MSM supplemented Müller-Hinton mediaor Müller-Hinton (plus NaCl) after 7 days MSM Concentration PMHS FMHSPMH FMH 16% 4.4 4.7 3.9 7.8 15% 4.7 4.7 3.8 7.9 14% 4.9 4.7 3.3 7.6 13%3.9 4.4 3.2 7.2 12% 6.0 4.1 6.0 6.5 11% 6.8 6.2 6.6 7.0 10% 6.8 6.9 7.07.0  9% 6.9 7.0 7.0 6.5  8% 7.2 7.0 7.8 7.2  7% 7.5 6.8 6.7 7.6  6% 8.08.0 7.5 7.6  5% 8.0 8.1 8.0 7.8

Example 6 Bactericidal Effect of MSM in Low Protein and SodiumChloride-Free Media

This example shows the bactericidal effect of MSM in low protein andsodium chloride-free media.

Lactose broth, free of both NaCl and of proteins, was used as the mediain this experiment. MSM was added to lactose broth in concentrationsranging from 5-16%. A duplicate set of MSM-containing media weresupplemented with DMSO to a final concentration of 1%. Each mediacomposition was initially inoculated with 6.75×10⁶ cfu/mL of E. coli.Cultures were incubated at 25° C. for seven days. Aliquots of eachculture were taken after 24 hours of culturing and at the end of sevendays in culture. Aliquots were serially diluted (with Modified Letheendiluent) and plated on trypto-soy agar plates. Plated cultures weregrown for 24 hours at 35° C. and then analyzed. Media compositions werecoded as shown in Table 6-1. Results of these studies are shown inTables 6-2 and 6-3.

TABLE 6-1 Media compositions Media Code Preparation LBM MSM added tolactose broth LBMD MSM added to lactose broth supplemented with 1% DMSOLB Lactose broth without MSM, spiked with E. coli. (−)LB negativecontrol (no E. coli.)

Lactose broth containing from 11-16% MSM reduced bacterial growth fromabout 1 log (16% MSM) to a maximum of about 2.2 logs (11% MSM) as shownin Table 6-2. Inhibition of bacterial growth was reduced by 1 log ormore from 9-16% MSM.

TABLE 6-2 Log of 24 hour E. coli growth in MSM- lactose broth with orwithout DMSO MSM Concentration LBM LBMD 16% 5.8 5.3 15% 5.6 5.5 14% 5.55.3 13% 5.1 5 12% 5.5 4.9 11% 4.6 5.6 10% 7 4.8  9% 7.1 5.7  8% 8 7.8 7% 8 9.2  6% 8.1 8.4  5% 8.4 8.8

After 7 days of culturing, a more defined pattern of bacterial growthinhibition was evident (Table 6-3). 10% MSM in lactose broth held the E.coli population approximately equivalent to the initial inoculum.

TABLE 6-3 Log of 7-day E. coli growth in MSM- lactose broth with orwithout DMSO MSM Concentration LBM LBMD 16% 4 4 15% 3.9 3.6 14% 4 3.613% 3.3 3.2 12% 3.4 2.9 11% 2.5 3.1 10% 6.9 4.2  9% 7.9 7.9  8% 8.2 8.4 7% 8.4 8.6  6% 8.4 8.6  5% 8.5 8.6

Example 7 Evaluation of Bactericidal Effect of MSM in Cosmetics

This example shows the bactericidal effect of MSM in cosmetics.

An initial evaluation of the bactericidal effect of MSM in a cosmeticmatrix was performed. The cosmetic matrix was a cream base (jojoba) thatis used in many cosmetic products. MSM was incorporated into the creamat concentrations ranging from 5-16% MSM. Each of these concentrationswas then spiked with E. coli at a level of 4.6×10⁵ cfu/mL and incubatedat 25° C. for 48 hours. After 48 hours, aliquots of each culture werediluted and plated on tryptic-soy agar, which was then incubated at 35°C. for 24 hours before counting. The results of these studies are shownin Table 7-1.

TABLE 7-1 Log of 48 hour E. coli growth in MSM-containing cosmeticmatrix MSM Concentration Cream 16% 1 15% 1 14% 1 13% 1 12% 1 11% 0.7810% 1.5  9% 1.9  8% 1.8  7% 2.5  6% 2  5% 3.16

These data indicate that bacteria growing in a cosmetic cream areparticularly sensitive to MSM. Surprisingly, lower concentrations of MSM(e.g., the 5-9% concentration range) substantially inhibited bacterialgrowth in this study. Thus, in several embodiments, MSM inconcentrations greater than 5% is used to inhibit microbial activity.

Example 8 Evaluation of Bactericidal Activity of 10% MSM in a CosmeticBase with or without Preservative Over 28 Days

This example shows the bactericidal activity of 10% MSM in a cosmeticbase with or without preservative over a 28 day period.

To evaluate the ability of MSM to function as a long term antimicrobialin a cosmetic base, 10% MSM was incorporated into a cosmetic creammatrix spiked with E. coli, which was evaluated for a 28 day time periodusing the USP <51> AET protocol. The cosmetic cream matrix into whichthe MSM was incorporated was preservative free. An additional cream,with a preservative, was also spiked with E. coli and evaluated. Theresults of these studies are shown in Table 8-1 through 8-4.

TABLE 8-1 Effect of 10% MSM on Microbial Growth in a Cosmetic CreamWithout Preservative Test Organism Initial Inoculum 48 hrs 7 days 14days 28 days Aspergillus Niger 1.1 × 10⁵ 8 × 10³ 8 × 10³ 6 × 10³ 5 × 10²Candida Albicans 2.1 × 10⁵ <10 <10 <10 <10 Escherichia coli 4.8 × 10⁶<10 <10 <10 <10 Pseudomonas aeruginosa 1.89 × 10⁶  <10 <10 <10 <10Staphylococcus aureus 4.0 × 10⁶ <10 <10 <10 <10

TABLE 8-2 Log Reduction from Initial Microorganism Inoculum with 10% MSMin Preservative-free Jojoba Cosmetic Cream dilution of MSM Test Organism14 Days 28 Days Aspergillus Niger 1.2 2.3 Candida Albicans 4.3 4.3Escherichia coli 5.7 5.7 Pseudomonas aeruginosa 5.3 5.3 Staphylococcusaureus 5.6 5.6

TABLE 8-3 Microbial Growth in a Cosmetic Cream Containing a PreservativeTest Organism Initial Inoculum 48 hrs 7 days 14 days 28 days AspergillusNiger 1.1 × 10⁵ 2 × 10⁶ 1.6 × 10² 18 × 10¹ 3 × 10¹ Candida Albicans 2.1× 10⁵ <10 <10 <10 <10 Escherichia coli 4.8 × 10⁶ <10 <10 <10 <10Pseudomonas aeruginosa 1.89 × 10⁶  <10 <10 <10 <10 Staphylococcus aureus4.0 × 10⁶ 3 × 10⁴ 1.6 × 10⁴ <10 <10

TABLE 8-4 Log Reduction from Initial Microorganism Inoculum in JojobaCosmetic Crème Containing a Preservative Test Organism 14 Days 28 DaysAspergillus Niger 2.7 3.5 Candida Albicans 4.3 4.3 Escherichia coli 5.75.7 Pseudomonas aeruginosa 5.3 5.3 Staphylococcus aureus 5.6 5.6

These studies show that a cosmetic cream base containing MSM iseffective in substantially inhibiting microbial growth over a period of28 days. Further, these studies illustrated that under some conditionsMSM is a more efficient antimicrobial agent than a standard cosmeticpreservative. For example, 10% MSM reduced the microbial load to agreater degree at 48 hours as compared to preservative containing cream.Moreover, S. aureus was reduced to nearly undetectable levels at 48hours in the MSM containing cream. In contrast, thepreservative-containing cream showed a modest bacterial population of3×10⁴ bacteria after 48 hours. Despite a less robust initial phase,preservative containing cream bacterial load was reduced to the sameextent by the end of the study as compared to the preservativecontaining cream.

Example 9 Evaluation of MSM Antimicrobial Activity in TwoPreservative-Free Cosmetic Compositions

This example describes MSM antimicrobial activity in twopreservative-free cosmetic compositions.

As described in Example 8, above, 10% MSM was incorporated into thecosmetic matrices, which were spiked with various initial inoculationsof microbes. In accordance with the USP <51> AET test, these spikedmicrobe cultures were incubated for 28 days, with samples removed at 48hours, 7 days, 14 days, and 28 days for plating and subsequent colonycounting. The results of these studies are shown in Tables 9-1 through9-4 below.

TABLE 9-1 Effect of 10% MSM on Microbial Growth in Preservative FreeCosmetic Composition #1 Test Organism Initial Inoculum 48 hrs 7 days 14days 28 days Aspergillus Niger 8.0 × 10⁵ 9.0 × 10³ 4.0 × 10³ 5.0 × 10¹<10 Candida Albicans 2.0 × 10⁶ 2.1 × 10³ <10 <10 <10 Escherichia coli5.8 × 10⁶ 5.4 × 10⁴ <10 <10 <10 Pseudomonas aeruginosa 5.7 × 10⁶ 7.3 ×10³ <10 <10 <10 Staphylococcus aureus 5.3 × 10⁶ 1.9 × 10⁴ <10 <10 <10

TABLE 9-2 Log Reduction from Initial Microorganism Inoculum with 10% MSMPreservative Free Cosmetic Composition #1 Test Organism 14 Days 28 DaysAspergillus Niger 4.2 4.9 Candida Albicans 5.3 5.3 Escherichia coli 5.85.8 Pseudomonas aeruginosa 5.8 5.8 Staphylococcus aureus 5.7 5.7

TABLE 9-3 Effect of 10% MSM on Microbial Growth in Preservative FreeCosmetic Composition #2 Test Organism Initial Inoculum 48 hrs 7 days 14days 28 days Aspergillus Niger 8.0 × 10⁵ 9.0 × 10³ 3.0 × 10³ 1.3 × 10³6.0 × 10¹ Candida Albicans 2.0 × 10⁶ 9.0 × 10² <10 <10 <10 Escherichiacoli 5.8 × 10⁶ 1.7 × 10⁵ <10 <10 <10 Pseudomonas aeruginosa 5.7 × 10⁶2.1 × 10³ <10 <10 <10 Staphylococcus aureus 5.3 × 10⁶ 1.5 × 10⁵ <10 <10<10

TABLE 9-4 Log Reduction from Initial Microorganism Inoculum with 10% MSMPreservative Free Cosmetic Composition #2 Test Organism 14 Days 28 DaysAspergillus Niger 2.8 5.1 Candida Albicans 5.3 5.3 Escherichia coli 5.85.8 Pseudomonas aeruginosa 5.8 5.8 Staphylococcus aureus 5.7 5.7

These studies show that MSM exhibited effective antimicrobial propertiesin the absence of a preservative.

Example 10 Selected Concentrations of MSM Supports Microbial Activity

This example shows that selected concentrations of MSM support microbialactivity.

Side-by-side growth studies in media fortified with MSM at aconcentration of 0, 0.04, 0.1, 0.2, 0.4, and 1% MSM were compared to thegrowth curve of various microorganisms to a 0% MSM concentration controlsample. Each organism (Lactobacillus rhamnosus, Lactobacillusacidophilus, and Bifidobacterium bifidum) was grown in MRS bacterialgrowth medium (broth) and plated on MRS agar at different timeintervals. Results are expressed in colony forming units per milliliter(cfu/mL).

For each test organism, 100 mL aliquots of MRS broth were prepared withthe respective concentration of MSM as outlined. Initially, a 1%, 0.4%,and 0.2% MSM (+/−0.01%) test solution was prepared by adding 1 g or 0.45g of MSM into 110 g of MRS broth and 0.20 g of MSM into 100 g,respectively. The 0.1% and 0.04% test concentrations were prepared bymaking a 1:10 dilution of the 1% and 0.4% test solutions. Once each setof test media was plated for sterility, they were inoculated at a levelof 100 μl of inoculum per 100 g or mLs of test broth (1:1000 inoculumdilutions) with each respective microorganism. All bacterial organismswere incubated at 35° C.+/−2° C. for the duration of the study.

All samples were plated on MRS agar at time 0, 12, 36, 48, 60 and 72hours (+/−45 minutes). All preparations and plating were conducted atroom temperature. All plating events were incubated at 35° C.+/−2° C.for at least 2 days or 3 days for Bifidobacterium. Test samples wereplated in triplicate at each test date and the averages are reported.The results of these studies are provided in Tables 10-1 through 10-3.

For Lactobacillus rhamnosus samples (Table 10-1), within the first 12hours all MSM samples had recovered at least 12% or more than the 0%control. The 0.2% and 1% concentrations were 41% and 47% higherrespectively within the first 12 hours. All test values were in line at24 hours before leveling off, cultures were highly turbid suggesting theorganism was headed into stationary phase. However, after leveling offslightly at 36 and 48 hours, the counts in the samples with MSMcontinued to rise whereas the 0% control started to drop.

TABLE 10-1 Growth of Lactobacillus rhamnosus 0% 0.04% 0.1% 0.4% Time(hours) MSM MSM MSM 0.2% MSM MSM 1% MSM 0 1.4 × 10⁶ 1.7 × 10⁶ 1.7 × 10⁶1.9 × 10⁶ 2.2 × 10⁶ 1.5 × 10⁶ 12 1.7 × 10⁷ 2.1 × 10⁷ 1.9 × 10⁷ 2.4 × 10⁷2.2 × 10⁷ 2.5 × 10⁷ 24 1.4 × 10⁹ 1.5 × 10⁹ 1.3 × 10⁹ 1.4 × 10⁹ 1.4 × 10⁹1.4 × 10⁹ 36 2.0 × 10⁹ 1.8 × 10⁹ 1.8 × 10⁹ 2.0 × 10⁹ 2.1 × 10⁹ 2.7 × 10⁹48 2.0 × 10⁹ 2.1 × 10⁹ 2.3 × 10⁹ 2.3 × 10⁹ 2.1 × 10⁹ 2.4 × 10⁹ 60 3.0 ×10⁹ 2.6 × 10⁹ 3.0 × 10⁹ 2.3 × 10⁹ 2.8 × 10⁹ 2.6 × 10⁹ 72 2.7 × 10⁹ 2.6 ×10⁹ 3.0 × 10⁹ 3.2 × 10⁹ 3.0 × 10⁹ 3.8 × 10⁹

These studies suggest that MSM concentrations of about 0.1% to about 1%enhance the growth/function of Lactobacillus rhamnosus, with microbiallevels ranging from 11% to 41% higher by the end of 72 hours.

For Lactobacillus acidophilus samples (Table 10-2), the 0.04% and 0.1%MSM concentrations were the first to yield growth, followed by 0.2% and0.4% MSM at 36 hours and the 1% MSM sample by 48 hours. No growth wasrecovered from the 0% control, suggesting MSM had a positive impact onthe recovery. Lower MSM concentrations revealed a shorter recovery timethan the higher concentrations of MSM. The 0.04% and 0.4% MSM samplesresulted in high levels of growth for Lactobacillus acidophilus. Thesestudies illustrate that MSM affects microbial metabolism in a mannerthat promotes microbial adaptability and recovery.

TABLE 10-2 Growth of Lactobacillus acidophilus Time (hours) 0% MSM 0.04%MSM 0.1% MSM 0.2% MSM 0.4% MSM 1% MSM 0 1.0 × 10³ 1.0 × 10³ 1.0 × 10³1.0 × 10³ 1.0 × 10³ 1.0 × 10³ 12 1.0 × 10³ 1.0 × 10³ 1.0 × 10³ 1.0 × 10³1.0 × 10³ 1.0 × 10³ 24 1.0 × 10³ 1.5 × 10⁴ 1.2 × 10⁴ 1.0 × 10⁵ 1.0 × 10⁵1.0 × 10⁵ 36 1.0 × 10³ 8.7 × 10⁷ 3.4 × 10⁷ 7.3 × 10⁵ 1.6 × 10⁸ 1.0 × 10³48 1.0 × 10³ 2.2 × 10⁸ 2.1 × 10⁸ 7.4 × 10⁶ 2.2 × 10⁸ 4.7 × 10⁴ 60 1.0 ×10⁵ 3.4 × 10⁸ 1.7 × 10⁸ 1.7 × 10⁷ 3.1 × 10⁸ 2.8 × 10⁷ 72 1.0 × 10⁵ 4.7 ×10⁸ 2.7 × 10⁸ 8.9 × 10⁶ 3.6 × 10⁸ 1.6 × 10⁸

Bifidobacterium bifidum, a common microbe used in probiotics, was alsotested. All Bifidobacterium samples were incubated under anaerobicconditions. Oxygen indicators were used to verify anaerobic conditionsbetween plating intervals for the Bifidobacterium test samples andplating events.

By 48 hours, the 0.04% and 0.2% MSM samples were 1 log higher than the0% MSM control. The 0.2% MSM sample had the highest level of growth forBifidobacterium bifidum, followed by the 0.04% MSM sample.

As observed with the Lactobacillus rhamnosus (Table 10-2), the 0.1% to1% MSM samples continued to grow while the control was headed into adownward stationary growth phase (Table 10-3). One and 2 log increasesof Bifidobacterium were observed with the 0.04% and 0.2% MSMconcentrations, respectively (Table 10-3).

TABLE 10-3 Growth of Bifidobacterium bifidum Time (hours) 0% MSM 0.04%MSM 0.1% MSM 0.2% MSM 0.4% MSM 1% MSM 0 8.7 × 10⁴ 6.6 × 10⁴ 7.0 × 10⁴7.0 × 10⁴ 1.1 × 10⁵ 6.8 × 10⁴ 12 2.0 × 10³ 3.5 × 10³ 4.4 × 10⁴ 3.1 × 10⁴2.0 × 10³ 2.3 × 10⁴ 24 1.4 × 10⁵ 1.2 × 10⁵ 1.3 × 10⁵ 1.3 × 10⁵ 1.0 × 10⁵2.0 × 10⁵ 36 2.4 × 10⁵ 2.3 × 10⁵ 2.0 × 10⁵ 2.3 × 10⁵ 2.3 × 10⁵ 4.0 × 10⁵48 2.6 × 10⁵ 5.2 × 10⁶ 2.3 × 10⁵ 5.8 × 10⁶ 4.0 × 10⁵ 3.1 × 10⁵ 60 3.3 ×10⁵ 3.4 × 10⁷ 4.7 × 10⁵ 4.8 × 10⁷ 8.7 × 10⁵ 3.7 × 10⁵ 72 1.6 × 10⁶ 8.0 ×10⁷ 5.3 × 10⁵ 1.2 × 10⁸ 1.2 × 10⁶ 5.7 × 10⁵

Also tested by observation were the general growth characteristics ofprobiotic organisms in MSM-supplemented and MSM-free media. Colony sizeof Bacillus coagulans grown on 0% media and 5% MSM-containing media werecompared (see Example 13 for detailed description).

Example 11 Evaluation of MSM Influence on Shelf-Life

This example describes MSM effect on the shelf-life of milk.

MSM as an additive has been shown to increase the growth and recovery ofbeneficial microorganisms in a product. This example examined whetherMSM modified the microorganisms that affect the shelf life stability ofa product based on microbial count. Milk which has a relative shortshelf life was used as the product to evaluate in this study. Milk withconcentrations of fat were analyzed to study the effects of how thesolid concentration in the product may affect the MSM. The standardshelf life for milk is 18 to 21 days, the study was carried out to 28days.

Shelf-life study on milk products fortified with MSM at the 0.0%, 0.5%,1.0%, 2.5%, 5.0% and 10% was conducted. The time intervals for platingof solutions in days were on day 0, 7, 14, 21, 24 and 28. The influencesof the percent solids on MSM concentrations at the following percentswere evaluated: 0.0%, 1.0%, 2%, 10.5% and 40%. The growth curves ofrecovered colony forming units per milliliter (cfu/mL) of microorganismswere compared between the MSM concentrations, with the 0% MSMconcentration as a sample control. The MSM stock powder was supplied byBergstrom Nutrition with certificate of analysis. The powder was themicroprill formula, lot #0806809, expiration date Oct. 31, 2013. Allmedia, water and stock MSM powder was sterility checked prior to thestudy. The working MSM concentrations were prepared from a single 10.0%MSM solution and were diluted accordingly with bottled milk to get thedesired final concentration of MSM. The product was supplied by a localmilk processing plant. The samples were collected and the study began onthe day of processing. The product included two bottles of each producttype for each MSM concentration for each day of analysis. Total bottlesfor one product type were 72 for the entire experiment. All bottles ofthe same product type came from one lot of production.

The microorganisms analyzed were the normal flora found in the productafter processing. The product samples were held at 4° C. during theduration of the study. All prep and plating was conducted at roomtemperature. Each concentration of MSM was performed in duplicate. Eachdilution was plated in duplicate for each time interval sampled. Tocapture the appropriate colonies per milliliter, each organism at eachtime interval was plated at three dilutions. All plates were incubatedat 37° C.±0.5° C. for 48 hours before examination. The appropriatedilution plate was used for enumeration and averaged for reporting. Theappropriate plate for enumeration contained between 25 and 250 cfu/mL.

The MSM stock sample and all media prepared with MSM were tested forbackground levels of microorganisms on Tryptic Soy agar. The MSM stockwas <10 cfu/g and all test media were negative in all instances prior toinoculation. All time intervals for plating included negative controlplates during pouring for quality control purposes. All of the negativecontrol plates were absent for microorganism growth. The results ofthese studies are shown in Tables 11.1-15.1 below.

TABLE 11.1 Log Growth of Nonfat Milk MSM Time (days) Conc. 0 7 14 21 2428 0% 1.50 2.05 0.94 4.40 1.44 2.20 0.5%   0.00 1.00 0.70 4.41 4.68 3.791% 0.74 0.70 0.00 4.56 1.28 2.02 2.5%   0.74 0.70 1.65 4.37 3.69 0.00 5%0.00 0.70 0.00 1.15 3.20 0.00 10%  0.92 0.70 0.59 0.00 0.00 0.00

TABLE 12.1 Log Growth of 1% Milk Fat MSM Time (days) Conc. 0 7 14 21 2428 0.0% 0.35 1.36 0.00 4.44 4.44 5.74 0.5% 1.05 0.50 0.70 4.37 3.27 6.091.0% 0.35 1.23 0.70 4.37 5.27 5.36 2.5% 0.00 0.00 0.00 4.28 4.28 2.535.0% 0.00 2.55 0.59 0.59 1.28 0.00 10.0% 0.70 0.00 0.50 0.85 0.50 0.00

TABLE 13.1 Log Growth of 2% Milk Fat MSM Time (days) Conc. 0 7 14 21 2428 0.0% 1.90 1.60 2.44 4.45 4.44 4.93 0.5% 1.26 1.59 2.09 4.43 3.27 5.481.0% 1.04 3.21 0.00 4.07 5.27 6.56 2.5% 1.06 0.00 0.70 2.96 4.28 5.815.0% 1.39 0.35 0.00 3.97 1.28 0.59 10.0% 0.35 0.50 0.70 1.97 0.50 1.66

TABLE 14.1 Log Growth of 10.5% Milk Fat MSM Time (days) Conc. 0 7 14 2124 28 0.0% 0.35 0.42 1.98 4.43 2.94 4.7 0.5% 0.00 1.09 0.00 4.36 2.733.84 1.0% 0.50 0.00 0.94 3.30 2.24 3.91 2.5% 0.35 0.35 0.00 2.34 3.141.84 5.0% 0.00 0.85 0.00 0.35 1.99 0.00 10.0% 1.00 0.85 0.35 2.14 0.350.00

TABLE 15.1 Log Growth of 40% Milk Fat MSM Time (days) Conc. 0 7 14 21 2428 0.0% 0.00 1.89 2.08 4.47 4.23 4.75 0.5% 0.00 0.35 2.32 4.46 4.02 1.951.0% 0.74 0.00 2.37 4.35 3.33 3.86 2.5% 0.00 0.00 0.00 2.16 3.79 0.955.0% 0.00 0.80 0.50 1.25 1.03 0.35 10.0% 0.00 0.00 1.95 4.45 0.85 0.35

When evaluating all milk without the MSM, there was a spike in thecounts at day 21. This is a standard spike typical with milk products.The increase in the normal flora reaches a 2 log point and there is astart to the degradation of the product. At 4 logs the product shelflife is questionable and the sensory factors make the productundesirable.

The evaluation study takes into consideration the nature of the productbeing used. The product was taken from one lot production day. Microbialcounts for a single lot from milk products can vary by 0.5 to 1.5 logs.Looking at the day 0 growth, the ranges for each product are within 1.5logs of each other.

Day 7 shows that for the control and the MSM concentrations there was aslight increase in the microbial counts. There was one microbial countthat was higher than the other for each sample, except for the 10.5%milkfat product. The 10.5% product microbial counts were all within 0.75logs of each other, (control and MSM concentrations). The 40% and nonfatmilk products had an increase in the control sample. While the 1% milkproduct had a spike in the 5% MSM sample and the 2% milk product had aspike in the 1% MSM sample.

On Day 14, the products show normal microbial counts and growth rates.No abnormal growth is seen in the products. The lower milk fat productsare within expected microbial load variabilities, when comparing theblank to the MSM concentrations. 40% milk product demonstrates a lowermicrobial count for the 5.00% and 2.50% MSM concentrations, while theblank and the other MSM concentrations are all within 0.40 logs inmicrobial counts. 10.5% milk product demonstrates the blank being 1 loghigher than the MSM concentrations, with the 1.00% and 10.0% MSM beingthe only two with a microbial count.

On Day 21, the products microbial counts separated out with regards tothe blank and MSM concentrations. The 1% and nonfat milk productsindicated that MSM in the higher concentrations (5.0% and 10.0%) sloweddown the growth of the normal flora. While the control and the lower MSMconcentrations microbial counts increased to 4 logs. In the 2% milkproduct, the 10.0% MSM concentration and the 2.50% MSM concentrationslowed the normal flora growth rate. The 0.50%, 1.00%, 5.00% MSM and theblank control microbial counts were at 4 logs. The 10.5% milk productshowed the 5.00% MSM at 0.35 logs, slowing the growth rate compared tothe control, which was at 4.43 logs. The 0.50% MSM concentration was at4.36 logs, 1.00% MSM was at 3.30 logs, 2.50% MSM was at 2.34 logs and10.0% MSM was at 2.14 logs. With the increase in MSM concentrations themicrobial counts decreased, with the exception of the 5.00% MSM. The 40%milk product microbial load was relatively equal in count for thecontrol, 0.5%, 1.0% and 10% MSM. The 2.5% MSM concentration was two logslower than the control at 2.16 logs, while the 5.0% MSM was lower by3.22 logs.

On Day 24, the control and the 1.0% MSM for the nonfat milk productdropped to around 1.3 logs, while the 0.50% MSM maintained microbialcounts. The 2.50% MSM microbial counts dropped, while the 5.0% MMSincreased. There was no growth observed in the 10.0% MSM. The 1% milkproduct had a microbial count decreased in the 0.50% MSM, increased inthe 1.00% MSM, and no alteration in the control and 2.5% MSM. The 5.0%MSM increased and the 10.0% MSM decreased. These two higher MSMconcentrations maintained a low microbial count. For the 2% milk productall MSM concentrations and control continued to increase in themicrobial load. The 10% MSM continued to lag behind in the microbialcount. The 10.5% milk product demonstrated a decrease in the control andthe MSM concentrations; 0.5%, 1.0%, and 10.0%. The 2.5% MSM and 5.0% MSMcontinued to grow. The 40% milk product showed a slight decrease ingrowth for the control, 5.0% MSM and the two lower MSM concentrations.2.5% MSM microbial counts increased at 24 hours, while the 10.0% MSM hada significant decrease in microbial growth. 5.0% MSM and 10.0% MSM wereat count close to the initial day 0 microbial loads.

On Day 28, the nonfat milk products were greater than 2 logs microbialload. The 0.5% MSM concentration was at 3.79 logs. The higherconcentrations of MSM, 2.50%, 5.0% and 10.0% were at no growth for day28. The 1% milk product shows an increase for the control, 0.5% and 1.0%MSM. The 2.5% MSM sample had a decrease in microbial load, while the5.0% and 10.0% MSM were at no growth. The 2% milk product had anincrease for the 1.0% MSM, a slight decrease for the control, 0.50% and2.5% MSM. The 5.0% MSM and 10.0% MSM decreased to 0.59 logs and 1.66logs respectively. The higher fat milk products, 10.5% and 40%, show thecontrol continuing to increase in microbial counts. Both products havethe 1.0% MSM increasing, while the 10.5% product also had the 0.5% MSMincrease in microbial counts, the 40% product had the 0.5% MSM decrease.The 2.5% MSM decreased in microbial load for both products. The 5.0% and10% for the 10.5% milk product were no growth. The 40% product had amicrobial count of 0.35 logs for the 5.0% and 10.0% MSM concentrations.

These studies indicate that use of MSM as an additive to milk does notadversely effect the shelf life of milk. In particular, at day 21 therewas no MSM concentration that had a microbial count higher than thecontrol. Moreover, these studies indicate that in certain milk products,a concentration of 5.0% MSM or 10.0% MSM actually held the microbialload significantly lower than the control. These studies suggest thatMSM at such concentrations can be used to increase the shelf-life of aproduct, such as milk.

Example 12 Acidophilus Milk and Bacillus coagulans Growth in SimulatedGastric Acid Supplemented with MSM

This example describes acidophilus milk and Bacillus coagulans growth insimulated gastric acid supplemented with MSM.

To analyze the effects of methylsulfonylmethane (MSM) on the growth ofprobiotic microorganisms in fortified with MSM in a simulated stomachfluid. Previous studies have shown that the addition of MSM to growthmedia, aid in the growth rate of microorganisms. The study will measurethe effect of probiotic growth fortified with MSM in a simulated gastricacid fluid.

Microbial growth studies were performed in the presence of 0%, 0.25%,2.0% and 5%. Time intervals for plating were pulled every 3 hours for 15hours, then at 24 and 48 hours. The growth curves of recovered colonyforming units per milliliter (cfu/mL) of the microorganisms werecompared between the MSM concentrations with the 0% MSM concentration asa sample control for each microorganism. The MSM stock powder wassupplied by Bergstrom Nutrition with certificate of analysis. The powderwas the microprill formula, lot #0806809, expiration date Oct. 31, 2013.All media and stock MSM powder was sterility checked prior to the study.The study was run on two organisms over a two week period. Themicroorganisms analyzed included Lactobacillus acidophilus milk andBacillus coagulans (15BB Lot #90BC004A1MZ supplied by Ganeden).

For the lactobacillus acidophilus milk, 11 mLs of milk with a count of81,000 cfu/mL was added to 99 mLs of simulated gastric acid. For theBacillus coagulans 1 gram of powder was added to 99 mL of a Tryptic SoyBroth (TSB) to get a count of 108,000 cfu per mL of Bacillus coagulans.Eleven milliliters of the Bacillus coagulans TSB was added to 99 mLs ofsimulated gastric acid. The working MSM concentrations were preparedfrom a single 5.0% MSM solution and were diluted accordingly with milkor TSB to get the desired final concentration of MSM. All solutions wereverified for sterility before proceeding with the study. The workingsimulated gastric acid was incubated at 35.0±0.2° C. during the study.The pH of the simulated gastric acid was at 1.2.

Lactobacillus acidophilus milk was inoculated on MRS agar at the timeslisted previously. Bacillus coagulans was inoculated on Tryptic Soy Agar(TSA) at the times listed previously. All prep and plating was conductedat room temperature. Each concentration of MSM in the simulated gastricacid was performed in duplicate. Each dilution for each organism wasplated in triplicate for each time interval sampled. To capture theappropriate colonies per milliliter, each organism at each time intervalwas plated at six different dilutions. All plates were incubated at 35°C.±0.5° C. for 72 hours for all organisms, except for Bacillus which wasincubated for 48 hours, before examination. The appropriate dilutionplate was used for enumeration and averaged for reporting. Theappropriate plate for enumeration contains between 25 and 250 cfu/mL.

The MSM stock sample and all media prepared with MSM were tested forbackground levels of microorganisms on MRS agar and TSA. The MSM stockwere <10 cfu/g and all test media were <1 cfu/mL in all instances priorto inoculation (see Table below). All time intervals for platingincluded negative control plates during pouring for quality controlpurposes. All of the negative control plates were clean formicroorganism growth. The results of these studies are provided inTables 16.1 through 18.2 below.

TABLE 16.1 Stock culture Numbers Control Prior to Test SampleInoculation Lactobacillus acidophilus Milk Bacillus coagulans cfu/mLinoculum 8.1 × 10⁴ 1.08 × 10⁵ Cfu added to 8.1 × 10⁵ 1.08 × 10⁶ 99 mLCfu/mL in media 8.1 × 10³ 1.08 × 10⁴ at time 0

The numbers control is derived from growth of specific organism onappropriate media. The inoculating liquids were plated for enumerationon the appropriate media. To capture the appropriate colonies permilliliter, each liquid was plated in triplicate at four differentdilutions. The appropriate dilution plate was used for enumeration andaveraged for reporting. The appropriate plate for enumeration containsbetween 25 and 250 cfu/mL.

TABLE 17.1 Log Growth of L. acidophilus in milk in duplicate MSMConcentration in percentage Time in (Hours) 0 0 0.25 0.25 2.5 2.5 5 5Log Growth 0 0 0 0 0 0 0 0 0.82 3 0 0 0 0 1.3 1 0 0.82 6 0 0 0.52 0.52 00 0.82 1 9 0 0 0.82 0 0.82 0 0.52 0.52 12 0 1.00 1.00 0 1.22 0.82 0.821.37 15 0.52 1.43 1.56 1.3 1.3 1.37 1.43 1.43 24 0.52 0.82 1.37 1.271.67 1.6 1.67 1.64 48 0 0 0 0 1.3 1.22 1.22 1.43

TABLE 17.2 Log Growth of L. acidophilus in milk average Time in MSMConcentration in percentage (Hours) 0 0.25 2.5 5 Log Growth 0 0 0 0 0.413 0 0 1.15 0.41 6 0 0.52 0 0.91 9 0 0.41 0.41 0.52 12 0.5 0.5 1.02 1.1015 0.98 1.43 1.34 1.43 24 0.67 1.32 1.64 1.66 48 0 0 1.26 1.33

Acidophilus milk placed in simulated gastric acid was aided by MSM inthe recovery of the Lactobacillus acidophilus. Initial recovery was lessthan the detection limit of the method. MSM at 5.0% had an initial logrecovery of 0.41. Hour 3 showed a spike in the recovery for the 2.5%MSM, will maintaining the 5.0% MSM growth log. Hour 6 the 0.25% MSM hasa recovery of 0.52 logs, 5.0% MSM showed a recovery of 0.91 logs. The2.5% MSM had a decrease in growth to non-detectable. At hour 9, therewas a significant detection for all concentrations of MSM. The 0%control is remained below the detectable limit. At hour 12, the controlgrew to 0.5 logs matching the 0.25% MSM. The 2.5% and 5.0% MSM sampleshad growth rates at 1.02 and 1.10 logs, respectively. Hour 15demonstrated continual growth with the MSM concentrations at 1.43, 1.34,and 1.43 logs. The 0% MSM control increased by 0.48 logs to 0.98 logs.The 0% MSM and 0.25% MSM at 24 hours decreased in growth by 0.31 logsand 0.11 logs, respectfully. The 0.25% MSM increased by 0.30 logs and5.0% MSM increased by 0.23 logs. At Hour 48, the 0.25% MSM and the 0%MSM control decreased to below detectable limits. The 2.5% MSM decreasedby 0.38 logs and the 5.0% MSM decreased by 0.33 logs.

TABLE 18.1 Log Growth of Bacillus coagulans in duplicate Time MSMConcentration in percentage (Hours) 0 0 0.25 0.25 2.5 2.5 5 5 Log Growth0 0 0.52 0.52 0 0 0 0.52 0 3 0 0 0.52 0 0 0 0 0 6 0 0 0.52 0.52 0 0 0 09 0 0 0 0 0 0 0 0 12 0 0 1.12 0.52 0 0 0 0 15 0 0 0 0 0 0 0.52 0 24 0 00 0 0 0 0 0 48 0 0 0 0 0 0 0 0

TABLE 18.2 Log Growth of Bacillus coagulans average Time MSMConcentration in percentage (Hours) 0 0.25 2.5 5 Log Growth 0 0 0.26 00.26 3 0 0.26 0 0 6 0 0.52 0 0 9 0 0 0 0 12 0 0.82 0 0 15 0 0 0 0.26 240 0 0 0 48 0 0 0 0

The initial recovery of Bacillus coagulans indicated no recovery. The0.25% and 5.0% MSM samples had an average of 0.26 logs however. A lowrecovery was seen throughout the study for 0.25% MSM and at hour 15 forthe 5.0% MSM. The recovery was too low to come to a conclusion about theGastric Acid study for Bacillus coagulans. It is possible that theinitial 3 hour exposure killed the organism.

These studies reveal that in regards to the Acidophilus milk there is apositive impact on bacterial growth with the milk containing MSM. In thefirst 3 to 6 hours there is a slight increase in the log phase growth ofeach L. acidophilus in the 0.25%, 2.5% and 5.0% MSM concentrations. Athour 9, there is a significant recovery of the L. acidophilus from themilk in the MSM concentrations versus the control. Hour 12 is when therewas the first indication of recovery of the L. acidophilus in thecontrol milk at 0.5 logs, matching the 0.25% MSM. MSM concentrations of2.5% and 5.0% are at a 1 log recovery growth rate. Hour 15 the controlmaximizes out its' growth at 0.98 logs. The 0.25% MSM concentration maxsout at 1.43 logs. 2.5% and 5.0% MSM maxs out at hour 24 at 1.64 and 1.66logs, respectively. Hour 24 shows a die off for the control and 0.25%MSM. At 48 hours the control and 0.25% MSM go below the detectable limitfor the method and the 2.5% and 5.0% MSM are still above a 1 log oforganism. MSM seems to be aiding this process by accelerating theadaptation and allows for the microorganisms to adapt quicker toenvironmental stressors.

MSM treated samples showed an increase in the log phase of growth. Thislog phase increase is seen easiest in the 5.0% MSM concentration. The5.0% MSM is 0.45 logs higher than the control at hour 15, which is themaximum growth recovery for the control. The 5.0% MSM reached a maximumof 1.66 logs or 0.68 logs higher than the control. This indicates asurvival rate of daughter cell at a higher percentage than the 0%control sample. Thus, suggesting that the environment with MSM isconducive to cell multiplication and survival.

MSM also affected the stationary phase and die off phase. The controlstationary phase was shorter than the stationary phase of the 2.5% and5.0% MSM. From hour 15 to 24, the samples not only maintained the growthrate, but continued to increase in logs by a minimum of 0.24 logs. Theseresults indicate that MSM as an additive allowed the L. acidophilus tothrive longer, allowing the organism to establish itself for a betterhealth benefit. The control was nondetectable at hour 48. The 2.5% and5.0% MSM growth was still above 1 log. This indicates that survivabilityof the L. acidophilus in milk was greater with the MSM additive.

The Bacillus coagulans study indicated no recovery. This was possiblydue to the time exposure in the gastric fluid. A shorter time ofexposure would be beneficial for the survivability of the Bacilluscoagulans. The difference between the Lactobacillus acidophilus studyand the Bacillus coagulans was the matrix. Milk provided enough of abuffer to allow the survival of the L. acidophilus in the gastric fluid.

Example 13 Measure of Viability of Bacillus coagulans Supplemented withMSM

This example describes the effect of MSM on Bacillus coagulans viabilityand colony formation.

Microbial robust colony formation studies were conducted in the presenceof 0%, 1.0%, 2.0% and 5% MSM. The microorganisms were grown for 72 hoursin 30 milliliters of tryptic soy broth. At the end of the 72 hours, thebroth was measured for colony formation, with photographic documentationof colony formation on tryptic soy agar. Percent transmittance was alsomeasured on a 25 mm×25 mm area of the tryptic soy agar placed betweentwo microscope slides in a spectrophotometer.

The growth curves of recovered colony forming units per milliliter(cfu/mL) of the microorganisms were compared between the MSMconcentrations with the 0% MSM concentration as a sample control foreach microorganism. The MSM stock powder was supplied by BergstromNutrition with certificate of analysis. The powder was the microprillformula, lot #0806809, expiration date Oct. 31, 2013. All media andstock MSM powder was sterility checked prior to the study. Themicroorganisms analyzed included Bacillus coagulans 9BB Lot #0109E002supplied by Ganeden.

For the Bacillus coagulans, the organism was isolated and grown for 24hours before harvesting. The harvested microorganism was placed into asterile 100 mL bottle called dilution A. Dilution A was further dilutedinto one working solution, with a count of 210 Bacillus coagulans per 1mL, called dilution B. One milliliter of Dilution B was used toinoculate the 30 mL of TSB concentrations stated above. The working MSMconcentrations were prepared from a single 5.0% MSM solution and werediluted accordingly with TSB to get the desired final concentration ofMSM. All solutions were verified for sterility before proceeding withthe study.

Bacillus coagulans was inoculated on Tryptic Soy Agar (TSA) at 35°C.±0.5° C. for 72 hours for colony verification and population density.All prep and plating was conducted at room temperature. Eachconcentration of MSM in the study was performed in duplicate. Eachdilution for the microorganism was plated in triplicate for each sample.To capture the appropriate colonies per milliliter, the microorganismwas plated at six different dilutions. All plates were incubated at 35°C.±0.5° C. for 48 hours for the microorganism. The appropriate dilutionplate was used for enumeration and averaged for reporting. Theappropriate plate for enumeration contained between 25 and 250 cfu/mL.

The MSM stock sample and all media prepared with MSM were tested forbackground levels of microorganisms on MRS agar and TSA. The MSM stockwere <10 cfu/g and all test media were <1 cfu/mL in all instances priorto inoculation. All time intervals for plating included negative controlplates during pouring for quality control purposes. All of the negativecontrol plates were clean for microorganism growth. The results of thesestudies are provided below.

TABLE 19.1 Stock culture Numbers Control Prior to Test SampleInoculation Bacillus coagulans cfu/mL inoculum 2.1 × 10⁴ Cfu added to2.1 × 10⁴ 99 mL Cfu/mL in media 2.1 × 10² at time 0

The numbers control is derived from growth of specific organism onappropriate media. The inoculating liquids were plated for enumerationon the appropriate media. To capture the appropriate colonies permilliliter, each liquid was plated in triplicate at four differentdilutions. The appropriate dilution plate was used for enumeration andaveraged for reporting. The appropriate plate for enumeration containsbetween 25 and 250 cfu/mL.

TABLE 20.1 Bacillus coagulans Population Table MSM ConcentrationPopulation Count in Percentage Average 0 4.3 × 10¹⁰ 1.0 1.01 × 10¹¹  2.53.3 × 10¹¹ 5.0 2.8 × 10¹¹

The population count was based on the dilution of the tryptic soy brothafter 72 hours and inoculation on to tryptic soy agar plates. The plateswere incubated for 48 hours and enumerated.

TABLE 21.1 Bacillus coagulans weight MSM Concentration Population Weightin Percentage Average 0 0.0586 1.0 0.1363 2.5 0.9828 5.0 0.1260

The tryptic soy broth after 72 hours was centrifuged down in a conicalvial. The supernatant was pulled off and the pellet was washed. Thecentrifuging and washing was repeated three times. At the end of thethird wash, the vials with pellet were weighed. Each vial was weighedempty and recorded. The corresponding vial weight was then subtractedfrom the ending weight of the pellet and vial, to get the weight of theBacillus coagulans population. A 25×25 mm section of agar was cut out ofeach plate at the end of the 72 hour period and placed between twomicroscope slides. The slides were sealed to prevent the agar plug fromslipping. The wavelength was set at 546 nm, and two empty slides wereused as a blank. When examining the plates, one Bacillus coagulanscolony was observed to be larger and more robust looking with the 5.0%MSM compared to the 0% MSM control. The weights indicated a heaviergrowth or a colony that was larger in size adding to the weight of thebiomass.

TABLE 22.1 Bacillus coagulans percent transmittance MSM Concentration inPercentage Percent Transmittance Agar 61.1% 0.0 52.3% 1.0 51.5% 2.549.8% 5.0 45.6%

The percent transmittance was used to indicate Bacillus coagulans colonysize in which a decrease in transmittance indicates an increase incolony size as the colony inhibited light from passing through the agar.The addition of 1 to 5% MSM appeared to cause a decrease in the percenttransmittance. It was observed that the percent transmittance result maybe influenced by the sample size, sample location and agar variables.

From visual observation, it was noted that the colonies were larger insize following treatment with MSM as compared to the blank control. Thecolonies also resulted in a higher weight total when measuring thebiomass of the MSM treated samples compared to the blank control. Thesetwo results combined with the measured percent transmittance indicatethat MSM as an additive (at certain concentrations) positivelyinfluences the viability, health and size of Bacillus coagulans.

Example 14 Effect of MSM on Lactobacillus acidophilus Growth andRecovery in a Simulated Intestinal Tract Environment

This example describes the effect of MSM on Lactobacillus acidophilusgrowth and recovery in a simulated intestinal tract environment.

Microbial growth studies fortified with MSM were conducted at thefollowing concentrations: 0%, 0.25%, 2.0% and 5%. The time intervals forplating of solutions in hours were 0, 3, 8, 24, 30, 36, 48, 54, 60 and72. The growth curves of recovered colony forming units per milliliter(cfu/mL) of the microorganisms were compared between the MSMconcentrations with the 0% MSM concentration as a sample control foreach microorganism. The MSM stock powder was supplied by BergstromNutrition with certificate of analysis. The powder was the microprillformula, lot #0806809, expiration date Oct. 31, 2013. All media, waterand stock MSM powder was sterility checked prior to the study. The pH ofthe simulated gastric acid was at 1.2. The pH of the simulatedintestinal fluid was 6.8. The microorganisms analyzed includedLactobacillus acidophilus ATCC #4356:

For the Lactobacillus acidophilus, bottled milk was used as the product.One milliliter of a 9 log solution of organism was placed into a 99 mLof bottled milk and mixed by hand shaking. This was repeated for eachconcentration of MSM. The suspension was enumerated for eachconcentration of MSM and is referred to as the starting inoculum. Tenmilliliters of each MSM concentration and Lactobacillus acidophilus milkwas placed into 90 mL of simulated gastric acid for 20 minutes. Thegastric acid was pre-warmed to 35° C. and kept at 35° C. for theduration of the 20 minutes. At the end of the 20 minute period, 10 mL ofthe simulated gastric acid, Lactobacillus acidophilus and milk mixturewas placed into 90 mL of simulated intestinal fluid. The simulatedintestinal fluid was pre-warmed to 35° C. and kept at 35° C. during theduration of the study. The working MSM concentrations were prepared froma single 5.0% MSM solution and were diluted accordingly with bottledmilk to get the desired final concentration of MSM. All solutions wereverified for sterility before proceeding with the study.

Lactobacillus acidophilus intestinal solution was inoculated on MRS agarat the times listed previously. All prep and plating was conducted atroom temperature. Each concentration of MSM was performed in duplicate.Each dilution for each organism was plated in triplicate for each timeinterval sampled. To capture the appropriate colonies per milliliter,each organism at each time interval was plated at four dilutions. Allplates were incubated at 37° C.±0.5° C. for 72 hours in a CO₂environment, before examination. The appropriate dilution plate was usedfor enumeration and averaged for reporting. The appropriate plate forenumeration contained between 25 and 250 cfu/mL.

The MSM stock sample and all media prepared with MSM were tested forbackground levels of microorganisms on MRS agar and Tryptic Soy agar.The MSM stock was <10 cfu/g and all test media were negative in allinstances prior to inoculation. All time intervals for plating includednegative control plates during pouring for quality control purposes. Allof the negative control plates were absent for microorganism growth. Theresults of these studies are provided in the below Tables.

TABLE 23.1 Log Growth of Lactobacillus acidophilus MSM concentrationTime 0% MSM 0.25% MSM 2.5% MSM 5.0% MSM Starting 6.83 6.86 6.86 7.02Inoculum 20 min Gastric <1.00 <1.00 <1.00 <1.00 Hour 3 1.33 1.30 1.461.62 Hour 8 2.22 2.18 2.30 2.33 Hour 24 4.47 4.40 5.94 6.39 Hour 30 4.104.66 5.39 6.63 Hour 36 4.00 4.38 5.32 8.65 Hour 48 4.59 8.03 11.17 10.17Hour 54 8.97 8.02 13.23 13.33 Hour 60 9.23 9.14 11.27 12.76 Hour 72 9.219.34 12.87 12.78

Reviewing the data there is a benefit with the addition of MSM to theproduct in the growth and recovery of Lactobacillus acidophilus.Comparing the 0% MSM control versus the 5.0% MSM there is a significantincrease in the log phase of growth with Lactobacillus acidophilus.Within the first 24 hours the 5.0% MSM was 1.81 logs higher than thecontrol. Over the next 12 hours the control, the 0.25% and 2.5%decreased. The 5.0% MSM continued to increase over the same time period.At the end of the time frame the 5.0% MSM was at 8.65 logs, 4.65 logshigher than the control. From hour 36 to hour 48 the MSM concentrationsgrew at a faster rate than the blank control. 0.25% MSM increased by3.65 logs and 2.5% MSM increased by 5.85 logs compared to 0.59 logs forthe 0.0% MSM. That trend changed in the next 8 hours with the controlincreasing by 4.38 logs. The 0.25% MSM decreased, while the 2.5% and5.0% MSM did not increase as significantly at the control, there was anincrease of 2.07 and 3.16 logs. From hour 60 to hour 72 the blankcontrol dropped while the MSM concentrations increased.

Analyzing the data, another 24 hour period of testing would have helpedto better predict a die off stage. At 72 hours the graph indicates theLactobacillus acidophilus reaching the stationary phase. With noindication of a die off stage, it is hard to predict if the MSMconcentration would extend life of the population longer than thecontrol. What we do see is a increase of growth rate, with a higherpopulation achieved with the 2.5% and 5.0% MSM. MSM as an additive tothe Lactobacillus acidophilus products would increase the probability ofthe organism establishing itself in the intestinal tract. More organismsfaster would increase the benefit of taking a probiotic.

With the addition of MSM there is a benefit in Lactobacillus acidophilusrecovery after a decrease if population growth. At hour 24 to hour 36there is a decrease in growth for the 0.25% MSM, 2.5% MSM and the 0.0%MSM. While the 0.25% MSM and 2.5% MSM recovered in twelve hours with asignificant growth rate increase, the 0.0% MSM took another twelve hoursto show a significant growth rate. For the 5.0% MSM there was nodecrease in recovery for this time frame, just a slight decrease ingrowth rate. The 5.0% MSM took only 6 hours to produce a substantialgrowth rate increase after the slight decrease in growth rate at hour24. This shows how the MSM influences the recovery time forLactobacillus acidophilus. Increasing the recovery time forLactobacillus acidophilus would be a benefit to helping it to establisha intestinal colony sooner, increasing health benefit.

The study needs to be extended to 96 hours and beyond to observe if MSMas an additive can extend the Lactobacillus acidophilus populationlonger. A population that can establish itself for a longer time in theintestinal tract would be added benefit to probiotic products and to thepeople who take them.

MSM as a supplement with Lactobacillus acidophilus helps the organism toestablish itself faster, grow at faster rate and reaching a higherpopulation. These attributes would benefit people taking Lactobacillusacidophilus as a probiotic.

Example 15 Effect of MSM on Grass Growth and Nutrient Value

This example describes the effect of MSM on grass growth and nutrientvalue of such grass.

The effect of MSM on grass growth and nutrient value was evaluated bymonitoring grass growth under the following conditions: (1) fertilizeralone; (2) MSM alone (OptiMSM® GNC—Lot#0922904, rate of 1:500 or 2 lbsper 1,000 sq. ft.); and (3) fertilizer and MSM (MSM at the rate of 1:500or 2 lbs per 1,000 sq. ft. in the presence of fertilizer) applied to thesame field but through a separate application. The fertilizer tested wasUrea (45-0-0) and type of pasture included the following mix of grasses(tall fescue, perennial rye grass, orchard grass, Timothy, white clover,medium red clover and intermediate rye grass; seeds for such formulationare commercially available on the World Wide Web at web addressoregroseeds.com/allnatdairy.html). The field to be tested was measuredand the marked to denote different levels of MSM application andcontrol. Using a controlled broadcast spreader, the MSM and/orfertilizer were applied. The field was irrigated as usual (sprinklerirrigated every four days). The grass was allowed to grow for sevenweeks and three days before being cut for testing. The sampled grass wascut one inch from the soil and placed in plastic bags for drying beforeshipment. The results of these tests on nutrient value are shown in theTable 23.2 below.

TABLE 23.2 1:500 Results on reported on a Dry basis MSM/Fert MSMFertilizer AOAC Percent Percent Percent Method Dry Matter 43.26 39.5744.62 934.02 Moisture 56.74 60.43 55.33 934.02 Crude Protein 20.03 24.3622.26 2001.11  Acid Detergent Fiber 30.95 25.77 29.44 973.18 NeutralDetergent Fiber 52.84 35.23 48.44 2002.04  Cell Solubles 47.16 64.7151.56 Calc Lignin 4.21 4.62 3.98 973.18 Ash 10.89 11.25 11.26 942.05Estimated TDN/DDM 64.47 68.39 65.61 Calculation Net Energy lact(Mcal/lb) 0.66 0.71 0.67 NFTA calc Est Net Energy (Mcal/lb) 0.55 0.60.57 NFTA calc Calcium 0.47 0.96 0.55 968.08 Phosphorus 0.46 0.43 0.42964.06 Magnesium 0.16 0.28 0.17 968.08 Potassium 3.97 3.48 4.09 968.08Sodium 0.03 0.3 0.05 983.04 Copper 14.57 8.82 8.18 968.08 Iron 94.51157.77 128.15 968.08 Zinc 25.75 24.79 25.76 968.08 Manganese 40.23 33.0833.73 968.08 Selenium 2.61 3.23 2.87 996.17 Quantitative Nitrate 0.30.05 0.64 968.07 Relative Feed Value 114.06 181.73 126.68 NFTA calc Est.RFV-Ash Corrected 191.08 132 Calculation Chloride 0.94 0.62 0.34 915.01Sulfur 0.32 0.25 0.36 923.01 Protein Solubility 46.48 59.52 52.56 923.04Non Structural Carbo 12.74 25.66 14.54 Calculation

Also, it was noted that while all of the grasses evaluated grew at equalrates, the rye grass grew 2 to 3″ taller in the MSM treated areas.Further, it was noted that there was no visible color variation betweenthe MSM and non-MSM treated grass. Moreover, it was noted that horsespreferred the MSM-treated pasture grass over the non-MSM treated grass.

These studies indicate that MSM can alter the nutrient value of grass(for example, it can increase the relative feed value as compared tofertilizer alone), possibly the flavoring of grass as well as grassheight depending upon the type of grass.

Example 16 Effect of 0.5% MSM on Fermentation Efficiency Related to theProduction of Beer (Scotch Ale)

This example describes the effect of 0.5% MSM on the fermentationefficiency related to the production of beer, in particular Scotch Ale.

It has been demonstrated herein that MSM at certain concentrations has apositive effect on microorganisms, including microorganism growth. Thispositive impact includes such organisms as fungi, yeast, and bacteria.Yeast cultures are involved in the production of beer during thefermentation process to produce ethanol and carbon dioxide. This studywas determined if 0.5% MSM by weight had a positive impact, such asincreasing efficiency of the brewing process. MSM was added to the YeastStarter (1000 mL H₂O, 100 g dried malt extract, 1 vial of white labsEdinburgh Ale Yeast) and to the wort. Wort is the liquid extracted fromthe mashing process during the brewing of beer or whisky. Wort containsthe sugars that are fermented by the brewing yeast to produce alcohol.

First, the Yeast Starter was prepared according to standard methodsknown to those of skill in the art except 0.5% MSM was added to atreatment group and no MSM added to a control group. This Procedure isdetailed below. Materials included the following: 2, 64 ounce glassjugs; funnel; 2-standard type brewing air-locks; 5.0 grams MSM; 2000 mLsof Water; 200 grams of Dried Malt Extract (DME); 2-vials of White LabsEdinburgh Ale WLPO28 yeast extract; and brewing sanitizer (San Star).

Preparation of Treatment Starter batch included the following steps: (1)glass jugs, airlocks and funnel were thoroughly cleaned and then rinsedwith beer sanitizer; (2) 1000 mLs of water was boiled then 100 grams ofDME was added; (3) sample was boiled for 10 minutes; (4) sample wasremoved from heat and 5.0 grams of MSM added; and (5) solution wasallowed to cool to 72° F. The Treatment Starter batch was then placed ina sanitized 64 ounce glass jug using to which 1-vial of White LabsEdinburgh Ale yeast was added. The airlock was applied and the entirevessel was placed in a dark room at room temperature for 48 hours.

Preparation of Control Starter Batch included the following steps: (1)glass jugs, airlocks and funnel were thoroughly cleaned and then rinsedwith beer sanitizer; (2) 1000 mLs of water was boiled then 100 grams ofDME was added; (3) sample was boiled for 10 minutes; (4) sample wasremoved from heat; and (5) solution was allowed to cool to 72° F. TheTreatment Starter batch was then placed in a sanitized 64 ounce glassjug using to which 1-vial of White Labs Edinburgh Ale yeast was added.The airlock was applied and the entire vessel was placed in a dark roomat room temperature for 48 hours.

The MSM Treatment Starter showed signs of activity (bubbling throughtrap) at approximately 2 hours after yeast was pitched. Control startershowed no sign of activity until approximately 10 hours after yeast waspitched.

On Brew Day (2-days after Yeast starter was made) the mash was prepared.Materials to prepare the mash included the following: 18 lb of American2-Row base grain; 3 lb of Crystal Malt 40 L specialty grain; 1 lbCara-Pils Malt specialty grain; and water. A mash tun (a vessel used inthe mashing process to convert the starches in crushed grains intosugars for fermentation) and Brew kettle were cleaned with powderedbrewers wash and rinsed thoroughly. The mash tun was then sanitized (SanStar Brewing Sanitizer). The following grains were crushed and milledfor mashing: 18 lb of American 2-Row base grain; 3 lb of Crystal Malt 40L specialty grain; and 1 lb Cara-Pils Malt specialty grain. Sevengallons of water were heated to 163° F. and then combined with preheatedmash tun. The crushed grains were then added and the solution was mixedthoroughly. The lid was attached and the solution allowed to mash for 60minutes. After 60 minutes, 4.25 gallons of work was drained from themash tun into the brew kettle. Strike water was preheated to 168° F.,added to the mash tun and mix thoroughly with grain. The mixture wasallowed to incubate for 10 minutes. This process repeated two more timesuntil a total pre-boil volume of 12.75 gallons was achieved in the brewkettle.

After preparing the mash, the brewing process was started. The followingmaterials were used for the brewing process: 3.0 oz Cascade hops; 2 tsp.of fish moss; 105 grams of MSM; brew kettle containing 12.75 gallons ofwort; wort chiller; 2 fermentation vessels; refractometer; brewingsanitizer; powdered brewers wash (PBW); and a filtered air stone.Equipment was cleaned thoroughly with PBW. The wort chiller and filteredair stone were sanitized with brewing sanitizer (San Star BrewingSanitizer). The wort (12.75 gallons) was brought to a boil in a brewkettle and a 1^(St) aliquot of Cascade hops (1.5 ounces) was added tothe solution. At 30 minutes of boil, a second aliquot (0.5 ounces) ofCascade hops was added. At 40 minutes of boil, third aliquot (0.5ounces) of Cascade hops was added as well as 2 teaspoons of fish Moss.At 50 minutes of boil, a fourth aliquot (0.5 ounces) of Cascade hops wasadded. Wort was decanted from the brew kettle to the wort chiller andchilled to 74° F. The wort was then divided into two fermentors (each 21liters in volume). 0.5% MSM (105 grams) was added to the treatmentfermentor. Brix reading of both fermentors was taken and the base pointwas recorded (Treatment fermentor=15 Brix; Control fermentor=14.75Brix). Each fermentor was Brix tested every 24 hours for 21 days. Bothfermentors were aerated for 25 minutes with sanitized filtered airstone. MSM infused yeast was pitched into treatment fermentor vesselwhile unaltered yeast was pitched into control fermentor vessel.Blow-off tubes were attached to both fermentors and fermentation wasallowed to proceed for 21 days. Results of these studies are provided inthe Table 23.3 below.

TABLE 23.3 MSM F.G. (Adjusted Control F.G. (Adjusted Day for Alc andTemp) for Alc and Temp) 1 1.026 1.028 2 1.018 1.020 3 1.016 1.018 41.016 1.017 5 1.015 1.017 6 1.015 1.015 7 1.015 1.015 8 1.015 1.015 91.015 1.015 10 1.015 1.015 11 1.015 1.015 12 1.015 1.015 13 1.015 1.01514 1.015 1.015 15 1.015 1.015 16 1.015 1.015 17 1.015 1.015 18 1.0151.015 19 1.015 1.015 20 1.015 1.015 21 1.015 1.015

When making a yeast starter the faster activation of the yeast culturetakes place the better for efficiency and for minimizing potentialenvironmental contamination from undesirable airborne micro-organisms.The MSM treated starter batch showed activity 80% sooner than thecontrol (2 hours compared to 10 hours). The study also indicated thatMSM aided in the fermentation process. Like the yeast starter, thefaster the activation of the yeast fermentation process the better forefficiency and for minimizing potential environmental contamination fromundesirable airborne microorganisms. The MSM treated fermentor showedactivity 58% sooner than the control (3.5 hours compared to 9 hours).The MSM treatment batch also reached maximum fermentation in 5 dayswhere the control batch took 6 days (a 17-25% sooner time ofcompletion).

These results indicate that MSM is useful in the process of beerbrewing.

Example 17 Growth of Lactobacillus acidophilus in Acidophilus MilkSupplemented with MSM

This example describes growth of Lactobacillus acidophilus inAcidophilus milk supplemented with MSM.

Microbial growth studies were conducted in Acidophilus milk fortifiedwith MSM at 0%, 0.5%, 2.5% and 5%. Time intervals for evaluation were at8 and 16 hours for a total of 104 hours. Then samples were evaluatedevery 7 days for a total of 28 days. The growth curves of recoveredcolony forming units per milliliter (cfu/mL) of the microorganisms werecompared between the Acidophilus milk with MSM concentrations with theAcidophilus milk with 0% MSM concentration as a sample control. The MSMstock powder was supplied by Bergstrom Nutrition with certificate ofanalysis. The powder was the microprill formula, lot #0806809,expiration date Oct. 31, 2013. The milks were bought at a local store.The Acidophilus milk was low fat (Darigold). The Acidophilus plusBifidus milk contained 2% milkfat (Lucerne). Acidophilus plus Bifidusmilk was run simultaneously with a MSM concentration of 2.5% and 0% as aproduct containing two microorganisms. The working solutions were heldat 4° C. during the study. The MSM milk working solutions were run induplicate.

All prep and plating was conducted at room temperature. All dilutionsfor all solutions were plated in triplicate for all time intervalssampled. To capture the appropriate colonies per milliliter, allorganisms at all time intervals were plated at three differentdilutions. All plates were incubated at 35° C.±0.5° C. in CO₂ for 72hours for all solutions. The appropriate dilution plate was used forenumeration and averaged for reporting. The appropriate plate forenumeration contains between 25 and 250 cfu/mL.

The MSM stock sample and all media prepared with MSM were tested forbackground levels of microorganisms on MRS agar and TSA. The MSM stockwere <10 cfu/g and all test media were <1 cfu/mL in all instances priorto inoculation. All time intervals for plating included negative controlplates during pouring for quality control purposes. All of the controlplates were clean for microorganism growth. At 72 hours, MSMconcentrations and negative control solutions were verified negative forcontamination. The results of these studies are provided in Table 24below.

TABLE 24 Log Growth of Lactobacillus acidophilus in Milk fortified withMSM MSM Concentration in percentage in acidophilus Milk Time Acido MilkA/B 0 A/B 2.5% 0.50% 0.50% 2.50% 2.50% 5% 5% 0 8.82 5.33 6.41 7.33 6.626.67 6.7 6.56 6.47 8 9.08 8.36 8.67 9.06 9.00 9.06 8.97 8.73 9.08 249.17 8.19 9.16 9.41 9.35 9.24 9.01 9.32 9.26 32 8.85 8.73 8.87 9.2 9.129.06 9.11 9.33 9.19 48 8.54 8.15 8.3 8.55 8.69 8.93 8.77 8.78 8.72 568.46 8.51 8.56 8.77 8.69 8.7 8.81 8.88 8.7 72 8.65 8.45 8.45 8.56 8.678.71 8.6 8.75 8.63 80 10.09 9.66 9.82 9.92 9.51 9.52 9.96 9.87 9.71 9610.02 9.68 8.61 9.93 10.25 10.18 10.31 10.14 10.26 104 9.45 8.83 8.929.47 9.52 9.8 9.45 9.74 9.38 Day 7  10.29 10.1 10.42 10.64 10.72 10.410.59 10.89 10.52 Day 14 6.96 6.83 5.70 6.81 5.63 8.00 6.66 7.82 8.26Day 21 5.30 5.60 5.37 5.37 5.37 5.56 5.37 5.64 5.48 Day 28 4.00 3.823.94 2.52 3.48 3.43 3.22 2.52 2.52

At hour 0 there was at least 1 log higher growth of Lactobacillusacidophilus in the milk without MSM compared to milk fortified with MSM.The significance is, at hour 8, the milk fortified with MSM showed aminimum of a 1.73 log increase in growth, while the milk without MSMshowed a 0.26 increase in growth rate. The MSM within the first 8 hoursof growth gave a significant increase in compared to the control. Thehighest increase in growth is the 5% MSM with an average log increase of2.39, while the 2.5% MSM was an increase average of 2.33 logs. Hour 24shows a growth rate that leveled off between control and the MSMconcentrations. The control had a 0.32 log decrease in growth at hour32. The MSM concentrations of 2.5% and 5% had a 0.04 and 0.03 logdecrease in growth, while the 0.5% was decreased by a 0.22 log at hour32. At hour 48 the control has a decrease of 0.31 logs, while the MSMdecrease is 0.54 logs for 0.5%, 0.24 logs for 2.5% and 0.51 logs for 5%.The MSM concentrations maintained a higher recovery rate compared to thecontrol. MSM concentration of 2.5% was on average 0.31 logs higher andthe 5% was 0.21 logs higher. Hour 56 showed no significant change ingrowth increase or decrease. Hour 72 the control increased by 0.19 logs,while the MSM concentrations were stable. Hour 80 had a significantincrease in growth. The control showed an increase of 1.44 logs. The MSMtreated samples showed an increase of growth at 0.5% (1.1 logs), 2.5%(1.09 logs), and 5% (1.1 logs). At Hour 96 the control stabilized. AllMSM concentrations increased on average (0.5%, 0.38 logs; 2.5%, 0.51logs; and 5.0%, 0.41 logs) for Hour 96. At Hour 96, the 2.5% MSMconcentration was 0.23 logs higher than the control. At Hour 104, thedecrease in log growth was comparable between the control and the MSMconcentrations (control decreased 0.57 logs; 0.5% MSM decreased 0.60logs; 2.5% MSM decreased 0.62 logs; and 5.0% MSM decreased 0.64 logs).Comparing final growth recovery between control and MSM concentrations,the study showed the 0.5% MSM at 0.05 logs higher than the control, the2.5% MSM at 0.18 logs higher than the control, and the 5% MSM at 0.11logs higher than the control.

Day 7 showed a growth increase from hour 104 for all working solutions.The control increased 0.84 logs, 0.5% MSM increased 1.19 logs, 2.5% MSMincreased 0.87 logs and 5.0% MSM increased 1.15 logs. 0.5% MSM was 0.39logs higher than the control, 2.5% MSM was 0.21 logs higher than thecontrol and 5.0% MSM was 0.42 logs higher than the control. Day 14showed a significant decrease in growth. The largest decrease in growthwas the 0.5% MSM at 4.46 logs. The control was next with a decrease of3.33 logs, 2.5% MSM at 3.17 and 5% MSM at 2.67 logs. 0.5% MSM was 0.74logs lower than the control, while the 2.5% MSM was 0.37 logs higher.The 5.0% MSM sample was a full log higher than the control at 1.08 logs.Day 21 continued the decrease in growth. The control was 1.66 logslower, 0.5% MSM was 0.85 logs lower, 2.5% was 1.87 lower and 5.0% MSMwas 2.48 logs lower. 0.5% MSM and the control were equal in log growth,with 2.5% MSM 0.17 logs higher than the control and 5.0% MSM 0.26 logshigher than the control. On Day 28, the decreased growth continued witha decrease of 1.3 logs for the control, 2.37 logs for 0.5% MSM, 2.14logs for 2.5% MSM and 3.04 logs for 5.0% MSM. The growth for thenegative control at Day 28 was 1.00 logs higher than 0.5% MSM, 0.68 logshigher than 2.5% MSM and 1.48 logs higher than 5.0% MSM.

The Acidophilus plus Bifidus milk over the course of the studydemonstrated similar growth rates. From Hour 0 to Hour 8 both showed asignificant increase in growth. At Hour 24, the control decreased 0.17logs, while the 2.5% MSM increased 0.49 logs, giving the 2.5% MSM a 0.97log higher count than the control. At Hour 32, the 2.5% MSM decreased0.29 logs and the control increased 0.54 logs, with the 2.5% MSM havinga 0.14 higher log count than the control. From Hour 48 to Hour 72 therewas a continual pattern of increase and decrease in growth, with the2.5% MSM having an increase growth of 0.15 and 0.5 logs over thecontrol. Hour 72 the growth was equal between the control and the 2.5%MSM. Hour 80 showed a growth increase of 1.21 logs for the control and1.37 logs for the 2.5% MSM, with the 2.5% MSM having a 0.16 log increasein growth. At Hour 96, there was a significant decrease in growth forthe 2.5% MSM of 1.21 logs. The control showed no significant differencefrom hour 80, resulting in a 1.07 log higher growth for the controlcompared to the 2.5% MSM. At Hour 104, the control growth decreased by0.85 logs and the 2.5% MSM samples increased by 0.31 logs. Hour 104showed the 2.5% MSM samples at 0.09 logs higher than the control. Day 7had an increase of 1.27 logs for the milk and 1.5 logs for the 2.5% MSM,with the 2.5% MSM being 0.32 logs higher than the milk Day 14 showed adecrease in growth, 3.27 logs for milk, 4.72 logs for 2.5% MSM. The milkhad a 1.13 increase in growth compared to the 2.5% MSM. Day 21 thedecrease slowed down, milk decreased by 1.23 logs and 2.5% MSM by 0.33logs, with the milk being 0.23 logs higher in growth than the MSM. Day28 milk decreased by 1.78 logs and 2.5% MSM decreased by 1.43 logs, withMSM being 0.12 logs higher than milk with no MSM. Table 25 displays thedata by averaging the duplicates.

TABLE 25 Growth of Lactobacillus acidophilus in Milk fortified with MSMaverage. MSM Concentration in percentage in acidophilus Milk Acido A/BTime Milk A/B 2.5% 0.50% 2.50% 5.00% 0 8.82 5.33 6.41 6.98 6.69 6.52 89.08 8.36 8.67 9.03 9.02 8.91 24 9.17 8.19 9.16 9.38 9.13 9.29 32 8.858.73 8.87 9.16 9.09 9.26 48 8.54 8.15 8.3 8.62 8.85 8.75 56 8.46 8.518.56 8.73 8.76 8.79 72 8.65 8.45 8.45 8.62 8.66 8.69 80 10.09 9.66 9.829.72 9.74 9.79 96 10.02 9.68 8.61 10.09 10.25 10.20 104 9.45 8.83 8.929.50 9.63 9.56 Day 7  10.29 10.1 10.42 10.68 10.50 10.71 Day 14 6.966.83 5.7 6.22 7.33 8.04 Day 21 5.3 5.6 5.37 5.37 5.465 5.56 Day 28 4.003.82 3.94 3.00 3.325 2.52

TABLE 26 Growth of non-probiotic microorganisms in Acidophilus Milk cfuper mL. MSM Concentration in percentage Day Acido Milk A/B 0 A/B 2.5%0.50% 0.50% 2.50% 2.50% 5% 5% 0 10 300 370 20 30 10 10 10 20 3 20 830480 10 10 30 10 10 10 7 10 1250 570 10 10 20 10 10 20 14 20 2500 1460 2020 <10 <10 20 10 21 20 8000 3500 20 40 60 170 20 10 28 650 120000 11000030 2250 2460 2700 10 10

Table 26 shows the data for standard plate counts analyzed on theworking solutions. This was performed to look at how MSM would affectthe normal flora found in the milks Day 0 was the day the samples weresetup for the start of the study. The Acidophilus plus Bifidus milkstarted with a higher count at Day 0 than what is typically expected.This caused the end values to be elevated. The Acidophilus milk productat Day 0 was at expected values. The Acidophilus milk maintainedadequate growth rates through out the study and were equivalent totypical growth rates seen in milk products. The 5.0% MSM did not allowany significant growth throughout the study.

MSM as an additive to this product played a significant role inincreasing the population of the probiotic, Lactobacillus acidophilus ina product. Within the first eight hours of fortifying a product withMSM, there was a significant influence on probiotic in the product.There was a significant increase in the growth rate of the probiotic.Acidophilus milk without MSM had a 0.26 long increase in growth withinthe first 8 hours. While the Acidophilus milk with MSM had a minimum of1.73 logs of growth. The 2.5% MSM milk sample had an increase of 2.27and 2.39 logs. The 5.0% MSM sample had an increase of 2.17 and 2.61logs.

Throughout the study there was a continual increase of growth whencomparing the Acidophilus milk control to the Acidophilus milk fortifiedwith MSM. The increase growth rate ranges from 0.04 to 1.08 logs overthe control. Only at two time points was there data that shows thecontrol growth higher than the MSM solutions, Hour 80 and Day 28. Whenanalyzing the data, the reason for the higher growth at Hour 80 was dueto the peak growth curve. Acidophilus milk without MSM peaked before themilk with MSM. Therefore, the MSM was still in the growth phase, whilethe Acidophilus milk had reached the peak of its growth. With theincrease in growth due to the MSM, there was a higher rate of die off atthe end of the study. Therefore, Day 28 showed a lower growth for theMSM solutions than the control.

Peak growth was reached with the Acidophilus milk fortified with 5.0%MSM, with a log of 10.89. Acidophilus Milk reached a peak growth of10.29 logs. Every concentration of MSM exceeded the growth of theAcidophilus milk 2.5% MSM had a peak growth of 10.59 logs and 0.5% MSMhad a peak growth rate of 10.72 logs. This further establishes theinfluence of MSM on a probiotic. The product once fortified with MSMexceeded the growth of the product without MSM. With the peak growthbeing higher, there was an increase growth seen at Day 14, a full weekpass the peak growth rate. The Acidophilus milk growth was 6.96 logs;the milk fortified with MSM was 7.82 and 8.26 logs, an increase of 0.86and 1.3 logs respectively.

Probiotic effectiveness is based on three points; Survivability,Colonization and Lactic Acid Production. MSM demonstrates the ability toaffect the survivability and colonization of the probiotic bacteria.Within the first eight hours the ability to colonize was seen with theincrease growth rate. At Day 14 the ability to survive was seen with theincrease log growth. The ability to increase Lactic Acid production wasthe third component of the effectiveness of a probiotic that will bestudied. In this study, there was an observed reaction of increased foamproduction in the MSM solutions.

The statement of MSM being a beneficial dietary supplement additive issupported by this study. Microbial flora of the gastrointestinal tractcan be impacted in a positive way with the addition of MSM in the humandiet. There was an increase in probiotic bacteria growth, with anincrease in survivability. Increasing the likelihood that MSM when usedin a probiotic product would increase the benefit to the consumer.

Example 18 Recovery of Lactobacillus acidophilus in Acidophilus MilkSupplemented with MSM

This example shows recovery of Lactobacillus acidophilus in acidophilusmilk supplemented with MSM.

To analyze the effects of MSM on the recovery of Lactobacillusacidophilus in Acidophilus milk, after a specified time of incubation adiluted portion of the original growth solutions were transferred to theappropriate broth and sampled at time intervals analyzing for recoveryrates. Microbial growth was determined in Acidophilus milk fortifiedwith MSM at 0%, 0.5%, 2.5% and 5%. At day 7, 14, 21 and 28 theAcidophilus milk samples fortified with MSM were diluted and transferredto the appropriate broths without MSM for the recovery study. Timeintervals for plating were pulled at every 24 hours over a 72 hourperiod. The growth curves of recovered colony forming units permilliliter (cfu/mL) of the microorganisms were compared between theAcidophilus milk with MSM concentrations with the Acidophilus milk with0% MSM concentration as a sample control. All media and stock MSM powderwas sterility checked prior to the study. The study was run on fourorganisms over a two week period. The microorganisms were split into tworuns, each a week long analyzing two microorganisms each week.

The Acidophilus milk was low fat (Darigold). The Acidophilus plusBifidus milk contained 2% milkfat (Lucerne). The Acidophilus Bifidusmilk was run simultaneously with a MSM concentration of 2.5% and 0% as aproduct containing two microorganisms. The working solutions were heldat 4° C. during the study. The MSM milk working solutions were run induplicate. All preparations and plating was conducted at roomtemperature. All dilutions for all solutions were plated in triplicatefor all time intervals sampled. To capture the appropriate colonies permilliliter, all organisms at all time intervals were plated at threedifferent dilutions. All plates were incubated at 35° C.±0.5° C. in CO₂for 72 hours for all solutions. The appropriate dilution plate was usedfor enumeration and averaged for reporting. The appropriate plate forenumeration contains between 25 and 250 cfu/mL.

The MSM stock sample and all media prepared with MSM were tested forbackground levels of microorganisms on MRS agar and TSA. The MSM stockwere <10 cfu/g and all test media were <1 cfu/mL in all instances priorto inoculation. All time intervals for plating included negative controlplates during pouring for quality control purposes. All of the controlplates were clean for microorganism growth. At 72 hours, MSMconcentrations and negative control solutions were verified negative forcontamination.

The study looks at the effect of MSM on the recovery of Lactobacillusacidophilus from Acidophilus milk fortified with MSM. The recovery studywas run parallel to the study done on the affects of MSM on growth ofLactobacillus acidophilus in Acidophilus milk fortified with MSM. Therecovery growth rate was analyzed on Day 7, Day 14, Day 21 and Day 28.For Table 27, the growth for Day x with no time is calculated from theinitial growth study with the dilution factor into the log growth. TheTable 28 growth was calculated from the log growth Day x with no timesubtracted from the log growth for the subsequential analyzed dates,e.g., Day 28 had a result of 4.00 logs, calculating the dilution the Day28 time 0 value is 2.00 logs for Table 27. Table 28 takes the value of2.00 as the starting value. Subsequential data for the hours analyzed,takes the counts in logs and subtracts the initial value of 2.00, e.g.,Day 28−24 is 11.29 logs, subtracting 2.00 logs the increase growth rateis 9.29 logs.

TABLE 27 Lag Recovery of Lactobacillus acidophilus in Milk fortifiedwith MSM Day 7 MSM Concentration in percentage Day-Hour 0% A/B 0% A/B2.5% 0.50% 0.5% 2.50% 2.5% 5% 5% Day 7-24 11.29 12.88 13.44 13.1 12.7813.16 13.15 12.12 13.29 Day 7-48 12.71 13.42 12.5 12.7 12.67 12.04 12.6713.09 13.25 Day 7-72 12.17 13.47 12.77 12.4 13.74 11.92 13.28 13.1 12.87

TABLE 28 Recovery of Lactobacillus acidophilus in Milk fortified withMSM Day 7 with average recovery by percentage of MSM with initial startdata MSM Concentration in percentage Acidophilus Ac/Bf Ac/Bf 0.5% 2.5%5.0% Day-Hour Milk Milk 2.5% MSM MSM MSM MSM Day 7 8.29 8.1 8.42 8.688.50 8.71 Day 7-24 11.29 12.88 13.44 12.94 13.16 12.71 Day 7-48 12.7113.42 12.50 12.69 12.36 13.17 Day 7-72 12.17 13.47 12.77 13.07 12.6012.99

Day 7 recovery shows that all MSM concentrations within the first 24hours of growth have a greater than 1 log increase over the control,0.5% MSM 1.65, 2.5% MSM 1.87, and 5.0% MSM 1.42. Hour 48, the controlwas slightly higher in growth compared to the 0.5% MSM at 0.03 logs, and0.36 logs higher than the 2.5% MSM, but 0.46 logs lower than the 5.0%MSM. Hour 72, the MSM concentrations exceeded the growth of the control;0.5% MSM at 0.9 logs, 2.5% MS at 0.43 logs and 5.0% at 0.82 logs.

Acidophilus plus bifidus control in the first 24 hours was at 12.88 logsfor growth, while the Acidophilus plus bifidus with 2.5% MSM was at13.44 logs growth. The 2.5% MSM milk was 0.56 logs higher than thecontrol. In the next two 24 hour periods the Acidophilus plus bifiduscontrol had growth at 13.42 logs and 13.47 logs. The Acidophilus plusbifidus with 2.5% MSM had growth at 12.50 logs and 12.77 logs over thesame time period. The control was 0.92 logs higher than the 2.5% MSM inthe second 24 hour period and was 0.7 logs higher in the third 24 hourperiod.

TABLE 29 Recovery of Lactobacillus acidophilus in Milk fortified withMSM Day 7 in log recovery growth rate increase from start time of zero.MSM Concentration in percentage Acidophilus Ac/Bf Ac/Bf 0.5% 2.5% 5.0%Hour Milk Milk 2.5% MSM MSM MSM MSM 0 0.00 0.00 0.00 0.00 0.00 0.00 243.00 4.78 5.02 4.26 4.66 4.00 48 1.42 0.54 −0.94 −0.26 −0.8 0.47 72−0.54 0.05 0.27 0.39 0.25 −0.19

Analyzing Day 7 recovery based on rate of growth increase in logs, therewas a significant increase within the first 24 hours of growth. Thecontrol increased by 3 logs from the initial inoculation, while the MSMconcentrations increased by 4.26 logs for the 0.5% MSM, 4.66 logs forthe 2.5% MSM, and 4.00 logs for the 5.0% MSM. In the second twenty foursthere was a decrease in the growth with respect to 0.5% MSM and 2.5%MSM. The control growth increased by 1.42 logs and the 5.0% MSM growthincreased by 0.47 logs. The third twenty four hour period, the controlgrowth decreased by 0.54 logs and the 5.0% MSM growth decreased by 0.19logs. The 0.5% MSM and 2.5% MSM increased in log growth, 0.39 and 0.25logs, respectively.

The Acidophilus plus bifidus milk control showed a 4.78 log increase inthe first 24 hours, compared to a 5.02 increase for the Acidophilus plusbifidus milk fortified with 2.5% MSM. The second 24 hour period theAcidophilus plus bifidus control increased by 0.54 logs, while the 2.5%MSM milk decreased by 0.94 logs. In the third 24 hour period, theAcidophilus plus bifidus control increased by 0.05 logs, while the 2.5%MSM milk increased by 0.27 logs.

TABLE 30 Lag Recovery of Lactobacillus acidophilus in Milk fortifiedwith MSM Day 14. MSM Concentration in percentage Day-Hour 0% A/B 0% A/B2.5 0.5% 0.5% 2.50% 2.50% 5.0% 5.0% Day 14-24 10.62 13.38 13.35 10.1910.10 10.00 9.73 9.6 9.6 Day 14-48 12.37 13.52 13.47 12.95 12.72 12.4312.2 12.99 13.04 Day 14-72 12.43 13.31 13.43 13.02 12.70 12.51 12.6312.65 12.21

TABLE 31 Recovery of Lactobacillus acidophilus in Milk fortified withMSM Day 14 with average recovery by percentage of MSM with initial startdata MSM Concentration in percentage Acidophilus Ac/Bf Ac/Bf 2.5% 0.5%2.5% 5.0% Day-Hour Milk Milk MSM MSM MSM MSM Day 14-0 4.96 4.83 3.704.22 5.33 6.04 Day 14-24 10.62 13.38 13.35 10.15 9.87 9.60 Day 14-4812.37 13.52 13.47 12.84 12.32 13.02 Day 14-72 12.43 13.31 13.43 12.8612.57 12.43

Day 14 the control exceeded the growth of the MSM concentrations in thefirst 24 hours. The control was 0.48 logs higher than the 0.5% MSM, 0.75logs higher than the 2.5% MSM and 1.02 logs higher than the 5.0% MSM.Hour 48 the control solution was 0.05 logs higher than the 2.5% MSM.0.5% MSM was 0.47 logs higher than the control and 5.0% MSM was 0.65logs higher than the control. Hour 72 the 0.5% MSM was 0.43 logs higher,2.5% MSM was 0.14 logs higher than the control and 5.0% MSM was equal tothe control.

Acidophilus plus bifidus fortified with 2.5% MSM was 0.03 logs lowerthan the Acidophilus plus bifidus control at hour 24. Hour 48 theAcidophilus plus bifidus control was 0.05 logs higher than theAcidophilus plus bifidus with 2.5% MSM. Hour 72 the Acidophilus plusbifidus with 2.5% MSM was 0.12 logs higher than the Acidophilus plusbifidus control.

TABLE 32 Recovery of Lactobacillus acidophilus in Milk fortified withMSM Day 14 in log recovery growth rate increase from start time of zero.MSM Concentration in percentage Acidophilus Ac/Bf Ac/Bf 0.5% 2.5% 5.0%Hour Milk Milk 2.5% MSM MSM MSM MSM 0 0.00 0.00 0.00 0.00 0.00 0.00 245.66 8.55 9.65 5.93 4.54 3.56 48 1.75 0.14 0.12 2.69 2.45 3.42 72 0.06−0.21 −0.04 0.03 0.26 −0.59

Analyzing Day 14 recovery based on rate of growth increase in logs, thefollowing was observed; the control increased by 5.66 logs from theinitial inoculation, while the MSM concentrations increased by 5.93 logsfor the 0.5% MSM, 4.54 logs for the 2.5% MSM, and 3.56 logs for the 5.0%MSM. In the second twenty fours, the control growth increased by 1.75logs while the 0.5% MSM increased by 2.69 logs, the 2.5% MSM increasedby 2.45 logs and the 5.0% MSM increased by 3.42 logs. The third twentyfour hour period, the control growth increased by 0.06 logs and the 5.0%MSM growth decreased by 0.59 logs. The 0.5% MSM and 2.5% MSM increasedin log growth, 0.0.03 and 0.26 logs respectively.

The Acidophilus plus bifidus milk control showed an 8.55 log increase inthe first 24 hours, compared to 9.65 increase for the Acidophilus plusbifidus milk fortified with 2.5% MSM. The second 24 hour period theAcidophilus plus bifidus control increased by 0.14 logs, while the 2.5%MSM milk decreased by 0.12 logs. In the third 24 hour period, theAcidophilus plus bifidus control decreased by 0.21 logs, while the 2.5%MSM milk decreased by 0.04 logs.

TABLE 33 Lag Recovery of Lactobacillus acidophilus in Milk fortifiedwith MSM Day 21 MSM Concentration in percentage Day-Hour 0% A/B 0% A/B2.5% 0.50% 0.50% 2.50% 2.50% 5% 5.00% Day 21-24 12.94 12.43 13.08 12.8912.93 10.62 10.95 9.52 9.85 Day 21-48 13.02 12.16 12.08 12.94 13.1 12.4413.12 13.08 13.07 Day 21-72 10.32 10.86 12.33 12.29 10.31 13.14 13.0513.26 13.04

TABLE 34 Recovery of Lactobacillus acidophilus in Milk fortified withMSM Day 21 with average recovery by percentage of MSM with initial startdata MSM Concentration in percentage Acidophilus Ac/Bf Ac/Bf 0.5% 2.5%5.0% Day-Hour Milk Milk 2.5% MSM MSM MSM MSM Day 21 3.30 3.60 3.37 3.373.47 3.56 Day 21-24 12.94 12.43 13.08 12.91 10.79 9.69 Day 21-48 13.0212.16 12.08 13.02 12.78 13.08 Day 21-72 10.32 10.86 12.33 11.30 13.1013.15

Day 21 the Acidophilus control in the first 24 hours had a log growth of12.94. 0.5% MSM the growth was 12.91 logs, 2.5% MSM was 10.79 logs and5.0% MSM was 9.69. The next 24 hour period the control was equal ingrowth to 0.5% MSM milk, 0.24 logs higher than the 2.5% MSM and 0.05logs lower than the 5.0% MSM. The final 24 hour period shows asignificant increase in the MSM concentrations compared to the control.0.5% MSM was 0.98 logs higher than the control, 2.5% MSM was 2.78 logshigher than the controla and the 5.0% MSM was 2.83 logs higher than thecontrol.

Acidophilus plus bifidus with 2.5% MSM was 0.65 logs higher than theAcidophilus plus bifidus control in the first 24 hours. Hour 48 theAcidophilus plus bifidus control was 0.08 logs higher. In the final 24hours the Acidophilus plus bifidus 2.5% MSM out grew the Acidophilusplus bifidus control by 1.47 logs.

TABLE 35 Recovery of Lactobacillus acidophilus in Milk fortified withMSM Day 21 in log recovery growth rate increase from start time of zero.MSM Concentration in percentage Acidophilus Ac/Bf Ac/Bf 0.5% 2.5% 5.0%Hour Milk Milk 2.5% MSM MSM MSM MSM 0 0.00 0.00 0.00 0.00 0.00 0.00 249.64 8.83 9.71 9.54 7.32 6.13 48 0.08 −0.27 −1.00 0.11 2.00 3.39 72−2.70 −1.30 0.25 −1.72 0.32 0.08

Reviewing the log increase for Day 21, in the first 24 hours theAcidophilus milk control had an increase of 9.64 logs. 0.5% MS had anincrease of 9.54 logs, 2.5% MSM had an increase of 7.32 logs and 5.0%MSM had an increase of 6.13 logs. In the second 24 hours the controlincreased by 0.08 logs. 0.5% MSM increased by 0.11 logs, 2.5% MSMincreased by 2.00, and 5.0% MSM increased by 3.39 logs. The final 24hours shows the control decreasing by 2.70 logs. 0.5% MSM decreased by1.72 logs. 2.5% MSM increased by 0.32 logs and 5.0% MSM increased by0.08 logs.

Acidophilus plus bifidus with 2.5% MSM increased by 9.71 logs and theAcidophilus plus bifidus control increased by 8.83 logs. In the finaltwo 24 hour periods the Acidophilus plus bifidus control decrease 0.27logs and 1.30 logs. The Acidophilus plus bifidus with 2.5% MSM decreased1.00 logs in the second 24 hour period and increased 0.25 logs in thefinal 24 hour period.

TABLE 36 Lag Recovery of Lactobacillus acidophilus in Milk fortifiedwith MSM Day 28 MSM Concentration in percentage Day-Hour 0% A/B 0 A/B2.5% 0.50% 0.50% 2.50% 2.50% 5.0% 5.0% Day 28-24 13.1 13.41 12.67 6.116.47 6.37 6.37 5.64 5.48 Day 28-48 11.04 13.42 13.19 13.66 12.99 12.5212.62 12.58 13.16 Day 28-72 12.47 10.31 12.00 12.99 13.01 12.98 12.3412.49 12.47

TABLE 37 Recovery of Lactobacillus acidophilus in Milk fortified withMSM Day 28 with average recovery by percentage of MSM with initial startdata MSM Concentration in percentage Acidophilus Ac/Bf Ac/Bf 0.5% 2.5%5.0% Day-Hour Milk Milk 2.5% MSM MSM MSM MSM Day 28 2.00 1.82 1.94 1.001.33 0.52 Day 28-24 13.10 13.41 12.67 6.29 6.37 5.56 Day 28-48 11.0413.42 13.19 13.33 12.57 12.87 Day 28-72 12.47 10.31 12.00 13.00 12.6612.48

Day 28 recovery data shows in the first 24 hour period the Acidophilusmilk control had a log growth of 13.10. The MSM concentrations were;6.29 logs for 0.5% MSM, 6.37 logs for 2.5% MSM, and 5.56 logs for 5.0%MSM. Hour 48 the 0.5% MSM log growth was 13.33, the 2.5% MSM was 12.57logs and the 5.0% MSM was 12.87 logs. The control at Hour 48 was 12.71logs. The control decreased to 12.17 logs at Hour 72. 0.5% MSM decreasedto 13.00 logs and 5.0% MSM decreased to 12.48 logs. 2.5% MSM improved to12.66 logs. This was a 0.53 log increase over the control.

Acidophilus plus bifidus control was 13.41 logs at Hour 24, 0.74 logshigher than the Acidophilus plus bifidus with 2.5% MSM. At Hour 48, thedifference was less with the control 0.23 logs higher than theAcidophilus plus bifidus with 2.5% MSM. At Hour 72, the Acidophilus plusbifidus with 2.5% MSM was 1.69 logs higher than the control, which wasat 10.31 logs.

TABLE 38 Recovery of Lactobacillus acidophilus in Milk fortified withMSM Day 28 in log recovery growth rate increase from start time of zero.MSM Concentration in percentage Acidophilus Ac/Bf Ac/Bf 0.5% 2.5% 5.0%Time Milk Milk 2.5% MSM MSM MSM MSM 0 0.00 0.00 0.00 0.00 0.00 0.00 2411.10 11.59 10.73 5.29 5.04 5.04 48 −2.06 0.01 0.52 7.04 6.20 7.31 721.43 −3.11 −1.19 −0.32 0.09 −0.39

At Day 28, growth rate increased in which the Acidophilus milk controlin the first 24 hour period increased 11.10 logs, 0.5% MSM increased5.29 logs, 2.5% MSM increased 5.04 logs, and 5.0% MSM increased 5.04logs. In the second 24 hour period, there was a shift to the controldecreasing by 2.06 logs, while the MSM fortified milk concentrationsincreased 0.5% by 7.04 logs, 2.5% by 6.20 logs, and 5.0% MSM by 7.31logs. The final 24 hour period showed the control increased by 1.43, the2.5% MSM increased by 0.09 logs, the 0.5% MSM decreased by 0.32 logs and5.0% MSM decreased by 0.39 logs.

Acidophilus plus bifidus milk control increased 11.59 logs in the first24 hours and the Acidophilus plus bifidus with 2.5% MSM increased 10.73logs. In the second 24 hour period, the Acidophilus plus bifidus controlincreased 0.01 logs and Acidophilus plus bifidus with 2.5% MSM increased0.52 logs. In the final 24 hour period, the Acidophilus plus bifiduscontrol decreased 3.11 logs, while the Acidophilus plus bifidus with2.5% MSM decreased 1.19 logs.

These studies show that MSM as an additive to this product played asignificant role in the recovery of the probiotic, Lactobacillusacidophilus. In every instance of recovery, there was an increase in thegrowth rate of Lactobacillus acidophilus with product fortified with MSMversus the product without MSM.

Day 7 recovery data showed in the first 24 hours MSM had a 0.99 log to1.66 log increase compared to the control. In the second 24 hour periodfor Day 7 even though the MSM growth rate was lower than the control,the overall growth numbers were higher for the 5.0% MSM, 0.46 logshigher. The third 24 hour period for Day 7, the MSM growth rate washigher than the control, by 0.36 logs, 0.79 logs and 0.93 logs.

At Day 14, only the 0.5% MSM out grew the control by 0.27 logs thegrowth rate in the first 24 hours. The 2.5% MSM and 5.0% MSM sampleswere 1.13 and 2.10 logs, respectively, lower than the control. This iswhere the control started to outgrow the MSM concentrations in the first24 hours. On Days 21 and 28, the control out grew all of the MSMconcentrations in the first 24 hours.

The second 24 hours for every data point that was collected after Day 7showed the MSM concentrations outperforming the control. Day 14 secondperiod data showed MSM growth rates at 0.70, 0.94, and 1.67 logs higherthan the control. Day 21 second period data showed MSM growth rates0.03, 1.92, and 3.31 logs higher than the control. Day 28 second perioddata showed MSM growth rates at 9.10, 8.26, and 9.37 logs higher thanthe control. This increase growth rate did not always translate to ahigher concentration of Lactobacillus acidophilus in the recovery broth.At Day 14, the 0.5% and 5.0% MSM concentrations were higher than thecontrol, while the 2.5% MSM was lower. Day 21 only the 5.0% MSM washigher. On Day 28, all three MSM concentrations were significantlyhigher than the control, by 2.29, 1.53, and 1.83 logs.

The third period data for Day 14 demonstrated that only the 2.5% MSMconcentration growth rate was greater, by 0.20 logs. Even with the lowergrowth rates, a higher log of growth was seen for the MSMconcentrations, except for 5.0% MSM which was equal to the control. OnDay 21, the third period data showed the MSM concentrations growth rateexceeding the control by 0.98, 3.02, and 2.78 logs. This growth rateincrease did translate to a higher concentration of Lactobacillusacidophilus for the MSM fortified samples. The growth recovery countswere 0.98, 2.78, and 2.83 logs higher than the control. The controlexceeded the MSM concentrations for Day 28's growth rate in the thirdperiod. Although the growth recovery counts for the MSM concentrationsof 0.5, 2.5 and 5% were 0.53, 0.19 and 0.01 logs, respectively, higherthan the control.

With growth curves of bacteria there is an initial lag phase where thebacteria adjust to the environment, before going into Exponential or logphase, where cells double. After the log phase there is a stationaryphase were growth rate slows. In this phase spikes and valleys are seenas growth slows. Finally, there is the death phase where bacteria runout of nutrients and die.

This study provides indicators of how MSM aids in the lag phase, logphase, stationary phase and death phase. MSM at different stagesshortens the lag phase, so that the probiotic bacteria begin the logphase at an earlier time. The log phase was extended beyond the controlin this study, so that the product with the MSM additive had a higherpeak value. The stationary phase was effected by the MSM as there was anextension of higher values for a longer period of time. The death ratewas slowed with MSM. At different points there was a slower rate ofdecline in growth. These different observations show that MSM as anadditive positively effects on probiotic bacteria. The benefit ofingesting a probiotic product fortified with MSM would be a fasterresponse time with a longer lasting effect. The consumer would get aproduct that increases their body responds to the added benefits ofprobiotic bacteria.

MSM consistently aided in the recovery and growth of probiotic bacteriain the product studied. Within the first 24 hours of growth, there wasan increase in rate of recovery indicating that stressed microorganismsresponded to a new environment better with MSM as an additive.

Example 19 Bifidobacterium bifidum Growth in Media Fortified with MSM

This example shows the effects of MSM on the growth of Bifidobacteriumbifidum in microbial growth media fortified with MSM.

Microbial growth studies were conducted in media fortified with MSM at0%, 0.125%, 0.25%, 0.5%, 1.0%, 2.5% and 5%. Time intervals for platingwere pulled every 8 hours for a total of 96 hours. The growth curves ofrecovered colony forming units per milliliter (cfu/mL) of themicroorganisms were compared between the MSM concentrations with the 0%MSM concentration as a sample control for each microorganism. The MSMstock powder was supplied by Bergstrom Nutrition with certificate ofanalysis. The powder was the microprill formula, lot #0806809,expiration date Oct. 31, 2013. All media and stock MSM powder wassterility checked prior to the study. The microorganism analyzed wasBifidobacterium bifidum ATCC #29521.

Bifidobacterium bifidum (99 mL of MRS broth with the addition of 0.05%L-cysteine) was prepared with respective MSM concentrations. The workingMSM concentrations were prepared from a single 5% MSM in MRS brothsolution and were diluted accordingly with MRS broth to get the desiredfinal concentration of MSM. The solutions were verified for sterilitybefore proceeding with the study.

The working solutions were inoculated at a level of 1.5 to 2 logs ofmicroorganism per 100 mL of broth. Bifidobacterium bifidum was incubatedunder anaerobic conditions at 35° C.±0.5° C. for 72 hours. Oxygenindicators were used to verify anaerobic conditions between platingintervals for the Bifidobacterium test samples.

Bifidobacteruim was inoculated on MRS agar supplemented with L-cysteineat the times listed previously to lower the oxidation-reductionpotential of media. All prep and plating was conducted at roomtemperature. All dilutions for all organisms were plated in triplicatefor all time intervals sampled. To capture the appropriate colonies permilliliter, all organisms at all time intervals were plated at sixdifferent dilutions. All plates were incubated at 35° C.±0.5° C. for 72hours. The appropriate dilution plate was used for enumeration andaveraged for reporting. The appropriate plate for enumeration containsbetween 25 and 250 cfu/mL. The MSM stock sample and all media preparedwith MSM were tested for background levels of microorganisms on MRS agarand TSA. The MSM stock were <10 cfu/g and all test media were <1 cfu/mLin all instances prior to inoculation. All time intervals for platingincluded negative control plates during pouring for quality controlpurposes. All of the negative control plates were clean formicroorganism growth. At 72 hours, MSM concentrations and controlsolutions were verified for negative for contamination of strains andthe strains were verified to original species.

TABLE 39 Stock culture Numbers Control Prior to Test Sample InoculationBifidobacterium bifidum cfu/mL inoculum 1.48 × 10⁴ Cfu added to 1.48 ×10⁴ 100 mL Cfu/mL in media 1.48 × 10² at time 0

The numbers control was derived from growth of specific organism onappropriate media. After incubation, the colonies were washed off themedia and captured in a sterile vial. The vial was used as the startingsolution for the number control (stock). The stock solution was thendiluted to get an appropriate reading on spectrophotometer, usingwavelength of 420 with percent light transmission. Bacterialconcentrations were determined according to AOAC Method 960.09, table960.09A. Preparation of Culture Suspension from Stock Culture wasDetermined by Spectrophotometer Reading or comparison to McFarlandstandard.

TABLE 40 Log Growth of Bifidobacteruim bifidum in Media Fortified withMSM. MSM Concentration in percentage Hr 0 0.125 0.25 0.5 1 2.5 5 0 1.221.12 1.00 1.00 1.00 1.30 1.12 8 2.55 2.89 2.93 2.84 2.73 2.71 2.04 167.16 7.85 8.07 7.45 7.73 7.56 6.10 24 8.94 9.06 8.49 8.81 8.81 8.64 8.4332 10.81 11.45 11.41 11.22 11.49 11.56 11.13 40 11.03 10.80 10.31 12.0911.19 11.73 11.78 48 11.54 8.22 8.10 9.39 10.43 10.72 10.70 56 10.647.25 7.22 9.51 10.57 11.34 10.59 64 9.97 8.86 6.77 10.34 10.66 12.2614.32 72 8.56 6.59 6.39 8.35 8.34 10.26 8.65 80 10.56 9.12 8.52 10.389.43 9.52 12.20 88 10.64 9.12 8.52 9.70 10.41 10.64 12.31 96 <6.00 <6.00<6.00 8.82 <6.00 <6.00 9.60

The growth observed with Bifidobacterium bifidum show a 0.2 to 0.4 logincrease in growth rate for the MSM concentrations of 0.125% to 2.5% athour 8. MSM concentrations of 0.125% to 2.5% at hour 16 increased to 0.3to 0.7 logs. Hour 24 showed the MSM concentrations of 0.125% to 2.5%slowing to be equal to or lower than the control. MSM at a concentrationof 5% showed slower growth rate compared to the control for the first 24hours. Hour 32 there was an increase in growth rate ranging from 0.3 to0.75 logs for all concentrations of MSM compared to the control. Hour 40the MSM concentrations of 0.125% and 0.25% showed a steady decline inrate of growth, to the extent that they were below the control from hour40 to hour 96. Hour 40 showed the 0.5% MSM sample at a full log higherin growth than the control. 0.5% MSM at hour 48 to hour 96 declined inthe growth rate to where it was 2 to 3 full logs below the control rateof growth. MSM at a concentration of 1% matched the growth rate of thegrowth from hour 40 to hour 96, except for hour 48 and hour 80 where itwas a full log less. 2.5% MSM at hour 40 was 0.7 logs higher in growthrate compared to the control. Hour 48 showed a 0.7 log decrease ingrowth rate compared to the control. Hour 56 to hour 72 2.5% MSM had agrowth rate that was 0.7 to 2.29 logs higher than the control. Hour 80showed a 1 log lower growth rate for 2.5% MSM sample compared to thecontrol and Hours 88 and 96 the growth rate was equivalent. 5% MSM athour 40 had a growth rate of 0.7 logs higher than the control. At Hour48, this dropped to 0.7 logs lower than the control and at Hour 56 thegrowth rate was equivalent to the control. Hour 64 shows a increasegrowth rate of 4.35 logs for the 5% MSM over the control. Hour 72 showeda decrease in growth rate, with a return to a 1.6 log increase in growthrate at hour 80 and hour 88. Hour 96 showed a 5% MSM rate of growth thatwas approximately 3.6 logs higher than the control.

Bifidobacterium bifidum showed a significant benefit to having MSM as anadditive to influence growth. All MSM concentrations increased thegrowth rate to the point where the Bifidobacterium bifidum reached inmaximum 16 hours before the control. The control reached a maximum of11.54 logs of growth at hour 48. This maximum growth was reached by allMSM concentrations at hour 32. MSM concentrations of 0.125% and 0.25%show a decline in growth from Hour 40 to Hour 96, never reaching themaximum growth again. 0.5% MSM increased the growth to 0.5 logs higherthan the maximum of the control. 0.5% MSM slowed the decrease of growthfrom hour 48 to hour 96. The 0.5% MSM delayed the die off stage to thepoint that at hour 96 there was 8.82 logs of growth, which wasapproximately 2 logs higher than the control. 1% MSM did not increasethe growth of the bacteria compared to the control, but it did decreasethe die off stage. From Hour 40 to Hour 64, the 1% MSM did not show alarge drop in growth, there was a slow drop of 0.5 logs for Hour 48, butthere was no decrease for Hours 56 and 64. Hour 72 there was a 2 logdrop in growth, but at Hour 80 there was a 1 log increase of growth andat Hour 88 there is another 1 log increase of growth. Hour 96 the growthwas outside the countable range and was estimated at less than 6 logs ofgrowth. Continuing for another 8 hours, there may have been anotherspike in the growth to over 6 logs. 2.5% MSM at hour 40 reached 11.73logs growth, with a drop of 1 log at hour 48. There was a steadyincrease of growth at Hour 56 and Hour 64, reaching a maximum of 12.26logs, 0.72 logs higher than the control. At Hour 72 there was a 2 logdrop, with a 0.7 log drop at Hour 80 for the 2.5% MSM. At Hour 88, the2.5% MSM increased the growth 1 log, before dropping below the countablerange at Hour 96. MSM at a 5% concentration was slower in increasing thegrowth rate compared to the other concentrations of MSM. At Hour 32, thegrowth was at 11.13 logs and Hour 40 the growth was 11.78 logs. At Hour48, the growth dropped 1 log and Hour 56 there was a 0.1 log drop. AtHour 64, the growth reached the highest for all the MSM concentrationsof 14.32 logs for the 5% MSM. There was a 6 log drop at hour 72, but athour 80 the growth increased 4 logs to 12.20. At Hour 88, a 0.1 logincrease was observed, before dropping to 9.60 logs of growth at Hour96. The 5% MSM slowed the death rate considerably, extending thestationary phase out 40 hours. Once the stationary phase was reachedthere was a continuing increase and decrease in growth, with movementtoward a lower growth pattern. These studies indicate that MSM isproceeding to the stationary phase faster for all concentrations,extending the stationary phase for concentrations over 0.5% MSM andincreasing maximum growth for the concentrations at 2.5% and 5% MSM.

Example 20 Effects of Bromcresol Purple on E. coli when MSM is Added tothe Matrix

This example shows the effects of Bromcresol Purple on E-coli when MSMis Added to the Matrix.

To investigate whether MSM functions as a carrier/transporter, theability of MSM to transport Bromcresol into the E. coli was evaluated.Bromcresol Purple is an indicator dye that turns yellow in the presenceof E. coli bacteria. It is non-toxic to the organism. To minimizepotential ionic interference Lactose Broth was chosen as the preferredmedia for this study because it is free of both NaCl and proteins.USP<51> Antimicrobial Effectiveness for testing was used as the templateto show the LC₁₀₀ (lethal concentration). MSM concentrations between5%-16% in increments of 1% were used. All concentrations were plated outto 10⁻⁷ dilutions to ascertain the log reduction.

Materials included the following: Lot number 0604751 of OptiMSM Flake;ATCC strain 8739 Escherichia coli lot: 57762704; 30 mL Borosilicateglass cultures tubes were used for all OptiMSM material; AccumediaMacConkey Broth (MB) Lot: 100,974A; Diluent used was Alpha BiosciencesModified Letheen Broth (MLB) Lot: 108-09; Alpha Biosciences Trypto SoyAgar with Lecithin; and Tween 80 (TSA) Lot: F08-42.

Flake OptiMSM was weighed out using a certified Mettler Toledo AG245balance SN: 1115210833 and aliquoted for each concentration. Thematerial was placed into 30 mL borosilicate glass culture tubes.Material was calculated on a 10 mL volume. Material was added to eachtube as follows: 5% (0.5 g), 6% (0.6 g), 7% (0.7 g), 8% (0.8 g), 9% (0.9g), 10% (1.0 g), 11% (1.1 g), 12% (1.2 g), 13% (1.3 g), 14% (1.4 g), 15%(1.5 g), and 16% (1.6 g). MacConkey Broth was aliquoted out in 10 mLs toeach tube then sterilize for 20 minutes at 121° C. Tubes were cooled toroom temperature which was approximately 20° C. All tubes were thenspiked with the same dilution of Escherichia coli that gave a level ofcolony forming units at 6.0×10⁶/mL (6.8). The tubes were then incubatedat 25° C. Daily observation for color change was performed during thefirst seven days. The tubes were mixed periodically to ensure that theOptiMSM was well equilibrated at all times. A positive and negativecontrol was ascertained.

The results of these studies are as follows:

(1) Day one: showed the color change of the broth to yellow forconcentration 5-7%; 8% showed slight clearing of color; and 9-16% showedno signs of change.

(2) Day two: showed same signs as day one.

(3) Day three: show a change in the 8% concentration turning to thetypical yellow color.

(4) Day 4 to day 6: showed no significant signs of change.

(5) Day 7: Showed the 9% turning to a yellow color. No color change from10%-16%.

(6) Day 14: Showed no signs for the concentration range for 10%-16%.

The concentrations tubes were streaked out on MacConkey agar to see iforganism could be recovered. No organisms were observed after the 72hour incubation. Day 30 showed no signs of change for the concentrationrange for 10-16%. Positive control was streaked for each time pointstreaked and showed signs of organism demonstrated by a classicalisolation streak.

This qualitative test indicates OptiMSM having some kind of carrieraffect and reducing or killing off the organism. This is demonstrated bythe lack of yellow color in MSM concentrations lower than what wasdemonstrated in previous studies using growth medium. Color showedreduction at concentrations as low as 8% versus 11% in the growth mediastudies.

Example 21 Antimicrobial Study of MSM and DMSO on StreptococcusOrganisms

This example shows the effects of MSM and DMSO on the growth ofStreptococcus organisms.

It has been demonstrated herein that specific concentrations of MSM(such as 10% to 16% MSM) kill microorganisms. Dimethyl sulfoxide hasalso been observed to kill microorganisms at concentrations of 30-50%.This study evaluated the bactericidal properties of both compounds,alone and in combination, as well as their efficacy when used with a lowlevel of penicillin.

Streptococcus pyogenes (Lancefield group A) has a hyaluronic acidcapsule and Streptococcus Pneumonia (no Lancefield Group identified todate) has a distinct polysaccharide capsule. These two organisms areresponsible for many types of human streptococcal infections and presenttwo different encapsulation types. Both of these organisms were used inthis in vitro study. In particular, this study determined theantimicrobial effects of MSM and DMSO, both individually and incombination, on Streptococcus pyogenes and Streptococcus Pneumonia. Thisstudy also determined the most effective concentrations forantimicrobial properties for both compounds and in combination andwhether combining MSM and DMSO reduces the concentrations of eithercompound needed to achieve microbial reduction. Further, theeffectiveness of using MSM, DMSO, and the combination of the two inconjunction with an antibiotic agent was evaluated.

Streptococcus Pneumonia (#10341™) and Streptococcus Pyogenes (Lancefieldgroup A, #10096™) were purchased from ATCC. MSM (#41631) and DMSO(#D8418) were purchased from Sigma-Aldrich. Penicillin was purchasedfrom Henry Schein. Bacterial culture medium was purchased fromBecton-Dickinson and company (#297963). The bioluminescent ATP assay kitwas purchased from Promega (#G8230). Streptococcus Pyogenes was culturedin Brain Heart Infusion broth (BD 237500, #44 booth) overnight. Equalamounts of bacterial containing broth were used for the studies.Streptococcus Pneumonia was also cultured in Brain Heart Infusion broth.

Bacterial Viability Evaluation:

The bioluminescent ATP assay kit was used to evaluate the bacterialviability, based on the following reaction:

ATP+D-Luciferin+O₂→Oxyluciferin+AMP+pyrophosphate+CO₂+light(560 nm).

Bacterial ATP can be measures by direct lysis of the bacteria with asuitable detergent; the released ATP is then free to react with theluciferin/luciferase and leading to the light emission. The intensity ofthe emitted light is proportional to the concentration of ATP.Measurement of the light intensity using a luminometer permits directquantitation of ATP, which is the universal indicator of viability forliving microorganisms.

Both S. pyogenes and S. pneumonia were cultured under various conditionsto determine optimal conditions to evaluate MSM, DMSO and/or PenicillinMSM, DMSO and Penicillin were diluted in culture medium according toTable 45-1. Bacterial was cultured for 7 hours for Streptococcuspneumonia and 18 hours for Streptococcus pyogenes respectively. Then thebacterial viability was evaluated by the bioluminescent ATP assay kit.The test was conducted in triplicates.

TABLE 41 Concentrations of MSM, DMSO and Penicillin evaluated. MSM (%)DMSO (%) Penicillin (μg/L) 20 20 100 10 10 50 5 5 25 2.5 2.5 12.5 1.251.25 6.25 0.625 0.625 3.125 0.3125 0.3125 1.5625 0 0 0

The MSM and DMSO were diluted in culture medium according to Table 42(for Streptococcus pneumonia, below left) and Table 43 (Streptococcuspyogenes, below right).

TABLE 42 DMSO (%) MSM (%) 0 0 5 10 20 5 0 5 10 20 10 0 5 10 20 20 0 5 1020

TABLE 43 DMSO (%) MSM (%) 0 0 2.5 5 10 2.5 0 2.5 5 10 5 0 2.5 5 10 8 02.5 5 10

To determination the effectiveness of using MSM, DMSO in conjunctionwith Penicillin, the MSM, DMSO and Penicillin were diluted in culturemedium according to Table 44-1 (S. pneumonia) and Table 44-2 (S.pyogenes).

TABLE 44-1 DMSO MSM Penicillin(μg/L) 5 5 25 10 20 10 5 10 20 20 5 10 20

TABLE 44-2 DMSO MSM Penicillin(μg/L) 2.5 2.5 3.125 5 10 2.5 6.25 5 10 52.5 3.125 6.25 8 3.125 6.25

The IC₅₀ of DMSO, MSM and Penicillin in Streptococcus pneumonia was12.86%, 15.97% and 68.54 g/L, respectively. DMSO and MSM had synergisticeffect within doses of 5% to 20% (for both drugs) in inhibitingStreptococcus pneumonia growth. DMSO and Penicillin also had synergisticeffect within doses of 10% to 20% (for DMSO) and 25 μg/L (forPenicillin) in inhibiting Streptococcus pneumonia growth. Further, MSMand Penicillin had a synergistic effect within doses of 5% (for MSM) and25 μg/L (for Penicillin) in inhibiting Streptococcus Pneumonia growth.When Penicillin, DMSO and MSM were used together, the greatestsynergistic effect resulted from DMSO+MSM only rather thanPenicillin+DMSO+MSM.

The IC₅₀ of DMSO, MSM and Penicillin in Streptococcus pyogenes was9.07%, 10.26% and 15.25 μg/L, respectively. DMSO and MSM had asynergistic effect within doses of 2.5% to 5% (for both drugs) ininhibiting Streptococcus Pyogenes growth. DMSO and Penicillin had asynergistic effect within doses of 5% (for DMSO) and 6.25 μg/L (forPenicillin) in inhibiting Streptococcus pyogenes growth. MSM andPenicillin had a synergistic effect within doses of 2.5% to 5% (for MSM)and 3.125 to 6.25 μg/L (for Penicillin) in inhibiting StreptococcusPyogenes growth. When Penicillin, DMSO and MSM were used together, thesynergistic effect resulted from DMSO+MSM only rather thanPenicillin+DMSO+MSM.

TABLE 45-1 Viability of S. pneumonia After DMSO Exposure DMSOConcentration Viability of S. pneumonia (%) 5 75.45 10 40.18 20 27.19

TABLE 45-2 Viability of S. pneumonia After MSM Exposure MSMConcentration Viability of S. pneumonia (%) 5 95.34 10 48.57 20 39.08

TABLE 45-3 Viability of S. pneumonia After Exposure to VariousConcentrations of MSM in 5% DMSO DMSO (%) MSM (%) S. pneumonia viability(%) 5 0 75.45 5 5 50.94* 5 10 45.40 5 20 27.22

TABLE 45-4 Viability of S. pneumonia After Exposure to VariousConcentrations of MSM in 10% DMSO DMSO (%) MSM (%) S. pneumoniaviability (%) 10 0 40.18 10 5 47.81 10 10 37.42 10 20 11.95

TABLE 45-5 Viability of S. pneumonia After Exposure to VariousConcentrations of MSM in 20% DMSO DMSO (%) MSM (%) S. pneumoniaviability (%) 20 0 27.19 20 5 17.60* 20 10 7.76 20 20 5.15

TABLE 45-6 S. pneumonia Viability After Exposure to VariousConcentrations of Penicillin Penicillin (μg/L) S. pneumonia viability(%) 25 79.82 50 42.70 100 40.93

TABLE 45-7 S. pneumonia Viability After Exposure to 25 μg/L ofPenicillin with Various Concentrations of DMSO Penicillin (μg/L) DMSO(%) S. pneumonia viability (%) 25 0 79.82 25 5 46.13 25 10 39.78 25 2022.08

TABLE 45-8 S. pneumonia Viability After Exposure to 50 μg/L ofPenicillin with Various Concentrations of DMSO Penicillin (μg/L) DMSO(%) S. pneumonia viability (%) 50 0 42.70 50 5 46.27 50 10 37.09 50 2019.14

TABLE 45-9 S. pneumonia Viability After Exposure to 100 μg/L ofPenicillin with Various Concentrations of DMSO Penicillin (μg/L) DMSO(%) S. pneumonia viability (%) 100 0 40.93 100 5 45.09 100 10 35.80 10020 21.76

The combination of 5% MSM with 25 μg/L of penicillin exhibited asynergistic reduction in the viability of S. pneumonia, leading to only41% viability (see Table 10). Synergy as compared to the expectedresults based on MSM alone and penicillin alone is indicated in theTables by an “*”. In contrast, 5% MSM alone reduced viability by onlyapproximately 5%, while 25 μg/L penicillin alone reduced viability byapproximately 21%. Thus, the 5% MSM/25 μg/L of penicillin combinationwas unexpectedly more efficacious than expected based on the resultsobtained with MSM or penicillin alone.

Moreover, as with DMSO, certain concentrations of MSM allowed lowerconcentrations of penicillin to reduce bacterial viability nearly aseffectively as higher concentrations. For example, 20% MSM with 100 μg/Lpenicillin reduced S. pneumonia viability to 21.37%, 20% MSM with 50μg/L penicillin reduced S. pneumonia viability to 20.75%. Thus, with useof 20% MSM, the required concentration of penicillin is reduced byone-half. Continuing this trend is the combination of 20% MSM with 25μg/L penicillin reduced S. pneumonia viability to approximately 25%.Similarly, though with a less robust reduction in bacterial viability,5% MSM allowed 25 μg/L penicillin to perform nearly identically to 100μg/L penicillin (compare Tables 45-10 through 45-12 for 25 μg/Lpenicillin)

TABLE 45-10 S. pneumonia Viability After Exposure to 25 μg/L ofPenicillin with Various Concentrations of MSM Penicillin (μg/L) MSM (%)S. pneumonia viability (%) 25 0 79.82 25 5 41.23* 25 10 41.83 25 2025.36

TABLE 45-11 S. pneumonia Viability After Exposure to 50 μg/L ofPenicillin with Various Concentrations of MSM Penicillin (μg/L) MSM (%)S. pneumonia viability (%) 50 0 42.70 50 5 41.23 50 10 47.47 50 20 20.75

TABLE 45-12 S. pneumonia Viability After Exposure to 100 μg/L ofPenicillin with Various Concentrations of MSM Penicillin (μg/L) MSM (%)S. pneumonia viability 100 0 40.93 100 5 41.75 100 10 36.67 100 20 21.37

Based on the synergistic results seen in certain combinations of MSM orDMSO with penicillin, the present study was performed in order toidentify the various combinations of MSM, DMSO, and penicillin thatyielded synergistic reductions in bacterial viability as compared tocombination the effects of combining DMSO, MSM, and penicillin onbacterial viability. This study was also designed to identifycombinations of the three compounds that advantageously allow one ormore of the compounds to be reduced, yet still efficaciously reducebacterial viability.

DMSO at 5, 10, and 20% was combined individually with MSM at one of 5,10, or 20% and penicillin at one of 25, 50, or 100 μg/L. Viability wasassessed as described above. Viability data are presented in Table45-13. The “*” symbol represents synergistic results as compared to thecorresponding combination of DMSO and penicillin. The “ψ” symbolrepresents synergistic results as compared to the correspondingcombination of MSM and penicillin. The values for the reduction inbacterial viability were added together to determine the thresholdreduction for synergy. For example, 5% DMSO reduces viability byapproximately 25% and 25 μg/L penicillin reduced viability byapproximately 21%, for a total combined reduction expected ofapproximately 46%. This represents 64% viability. Thus, if thecombination of 5% MSM, 5% DMSO, and 25 μg/L penicillin results in lessthan 64% viability, synergy between the compounds has been identified.

Several combinations of MSM, DMSO, and penicillin yield synergisticimprovements in bacterial reduction. For example, the combination of 5%DMSO, 5% MSM, and 25 μg/L penicillin reduced bacterial viability toapproximately 52% (see Table 45-13). Five percent DMSO in combinationwith 25 μg/L penicillin reduced bacteria viability to approximately 64%(e.g., about a 46% reduction, based on the individual reduction seenwith 5% DMSO, see Table 45-1, and the individual reduction seen with 25μg/L penicillin). Thus, the combination of all three compounds reducedbacterial viability by about an additional 12%. Similarly, thecombination of 5% MSM with 25 μg/L penicillin resulted in bacteriaviability of about 74%, while the combination of all three compoundsreduced viability by nearly an additional 22%.

In some combinations, synergistic results were detected with respect toboth DMSO and penicillin as well as MSM plus penicillin. For example,10% DMSO in combination with 20% MSM and 25 μg/L penicillin is yielded asynergistic improved in antimicrobial activity as compared to bothreference combinations. In other combinations, synergy was detected onlywith respect to either DMSO plus penicillin or MSM plus penicillin. Forexample, the combination of 5% MSM with 10% DMSO and 25 μg/L penicillinwas synergistic with respect to MSM plus penicillin, but not withrespect to DMSO plus penicillin.

In addition to the synergistic effects discussed above, there areseveral instances wherein the certain combinations of DMSO, MSM andpenicillin allow for a reduction in the efficacious concentration ofpenicillin. For example, as shown in Table 45-13, the combination of 5%DMSO with 20% MSM yields very similar overall bacterial viability overthe range of penicillin concentrations tested (from ˜25% viability with25 μg/L penicillin to ˜18% viability with 100 μg/L penicillin).Additionally, 10% DMSO with 20% MSM resulted in nearly identicalbacterial viabilities across the penicillin concentration range.

Similar results are seen with 20% DMSO in combination with 5, 10, or 20%MSM and any concentration of penicillin. These results reveal a slightlywider range of bacterial viability across the different penicillinconcentrations, however, given that the reduction in all casesapproaches approximately 90 to 95%, these combinations are all stilleffective.

TABLE 45-13 S. pneumonia Viability After Exposure to Variouscombinations of DMSO, MSM, and Penicillin DMSO (%) MSM (%) Penicillin(μg/L) S. pneumonia viability (%) 5 5 25 52.11*^(,ψ) 5 5 50 43.36 5 5100 53.03 5 10 25 51.82* 5 10 50 44.52 5 10 100 31.33 5 20 25 24.91* 520 50 19.20 5 20 100 18.12 10 5 25 44.41^(ψ) 10 5 50 38.24 10 5 10036.19 10 10 25 39.38 10 10 50 33.73 10 10 100 25.98 10 20 25 11.87*^(,ψ)10 20 50 11.03 10 20 100 10.96 20 5 25 12.74*^(,ψ) 20 5 50 13.39^(ψ) 205 100 9.28^(ψ) 20 10 25 7.69*^(,ψ) 20 10 50 7.74 20 10 100 5.58 20 20 254.93*^(,ψ) 20 20 50 5.60 20 20 100 1.80

As discussed above, the structure of S. pyogenes differs from that of S.pneumonia, and therefore additional experiments were undertaken toevaluate the synergistic effects of various concentrations of DMSO andMSM, as well as combinations of DMSO, MSM, and penicillin.

DMSO was added to S. pyogenes cultures to final concentrations of 0.31,0.63, 1.25, 2.50, 5.00, 10.0, or 20.0. At these concentrations, DMSOresulted in reductions in bacterial viability in a dose-dependentmanner. See Table 45-14. MSM alone was added to S. pyogenes cultures tofinal concentrations of 0.31, 0.63, 1.25, 2.50, 5.00, 10.0, or 20.0. Atthese concentrations, MSM also resulted in reductions in bacterialviability in a dose-dependent manner. See Table 45-15.

TABLE 45-14 Viability of S. pyogenes After DMSO Exposure DMSOConcentration Viability of S. pyogenes (%) 0.31 100 0.63 100 1.25 1002.50 100 5.00 96.66 10.0 14.50 20.0 5.14

TABLE 45-15 Viability of S. pyogenes After MSM Exposure MSMConcentration Viability of S. pyogenes (%) 0.31 100 0.63 100 1.25 1002.50 100 5.00 95.88 10.0 23.94 20.0 15.18

MSM and DMSO in combination were evaluated for their antibacterialeffects on S. pyogenes. DMSO at 2.5%, 5%, and 8% was combined with MSMat 0% (DMSO only control), 2.5%, 5%, and 10%. As shown in Tables 16, 17,and 18 certain combinations of MSM with DMSO are synergistic as comparedto the effects of either DMSO or MSM alone. Synergistic results ascompared to DMSO or MSM alone are indicated by an “*”. For example,addition of 2.5% MSM to 2.5% DMSO reduced bacterial viability toapproximately 65% (see Table 16), while the no effect of theseconcentrations of MSM and DMSO would be expected, as individually,neither compound reduced bacterial viability. The synergistic effect isalso seen with 2.5% DMSO and 5% MSM, where bacterial viability isreduced by nearly 83% (as compared to an expected 4% reduction based onthe compounds' effects alone). Synergy is also seen with 5% DMSO incombination with any concentration of MSM. Thus, in some embodiments,DMSO at 5% induces synergistic reductions in bacterial viability incombination with any concentration of MSM between 2.5% and 10%. In someembodiments, DMSO at 2.5% and MSM in concentrations between 2.5% and 5%are advantageously and unexpectedly synergistic at reducing bacteriaviability.

TABLE 45-16 Viability of S. pyogenes After Exposure to VariousConcentrations of MSM in 2.5 DMSO DMSO (%) MSM (%) S. pyogenes viability(%) 2.5 0 100 2.5 2.5 65.06* 2.5 5.0 17.71* 2.5 10.0 16.37

TABLE 45-17 Viability of S. pyogenes After Exposure to VariousConcentrations of MSM in 5 DMSO DMSO (%) MSM (%) S. pyogenes viability(%) 5.0 0 96.66 5.0 2.5 36.21* 5.0 5.0 7.87* 5.0 10.0 7.64*

TABLE 45-18 Viability of S. pyogenes After Exposure to VariousConcentrations of MSM in 8 DMSO DMSO (%) MSM (%) S. pyogenes viability(%) 8.0 0 9.96 8.0 2.5 14.37 8.0 5.0 5.97 8.0 10.0 5.60

Various concentrations of penicillin alone were evaluated for theirability to reduce viability of S. pyogenes. As shown in Table 45-19,penicillin decreased bacterial viability in a dose-dependent fashion.

TABLE 45-19 S pyogenes Viability After Exposure to VariousConcentrations of Penicillin Penicillin (μg/L) S. pyogenes viability (%)1.56 100 3.13 100 6.25 100 12.5 13.16 25.0 9.07 50 9.57 100 9.40

Due to the highly efficacious nature of penicillin concentrations at orabove 25 μg/L, DMSO was combined with concentrations of penicillin thatwere less efficacious (ranging from 3.125 to 12.5 μg/L). As such,identification of synergism between DMSO and penicillin would be lesslikely to be mathematically obscured.

As shown in Tables 45-20, 45-21, and 45-22 (identified by an “*”)several combinations of DMSO and penicillin resulted in synergisticresults. For example, 5% DMSO in combination with 3.125 μg/L penicillin,based on the efficacy of the two compounds alone, would only be expectedto reduce bacteria viability by about 4%. However, when combined, theactual reduction was approximately 10-fold greater (viability reduced to˜61%, see Table 45-20). Similar synergistic effects were seen when 5%DMSO was combined with 6.25 μg/L or 12.5 μg/L penicillin (see Table45-21 and 45-22, respectively).

TABLE 45-20 S pyogenes Viability After Exposure to 3.13 μg/L ofPenicillin with Various Concentrations of DMSO Penicillin (μg/L) DMSO(%) S. pyogenes viability (%) 3.13 0 100 3.13 2.5 100 3.13 5.0 60.85*3.13 8.0 12.90

TABLE 45-21 S pyogenes Viability After Exposure to 6.25 μg/L ofPenicillin with Various Concentrations of DMSO Penicillin (μg/L) DMSO(%) S. pyogenes viability (%) 6.25 0 100 6.25 2.5 100 6.25 5.0 60.23*6.25 8.0 6.91*

TABLE 45-22 S pyogenes Viability After Exposure to 12.5 μg/L ofPenicillin with Various Concentrations of DMSO Penicillin (μg/L) DMSO(%) S. pyogenes viability (%) 12.5 0 13.16 12.5 2.5 19.63 12.5 5.014.77* 12.5 8.0 6.43

Similar studies to those using DMSO were performed by combining MSM withpenicillin ranging from 3.125 to 12.5 μg/L. Results are shown in Tables45-23, 45-24, and 45-25. Synergy is indicated by an “*”. As with DMSO,previously ineffective concentrations of MSM and penicillin wereeffective in combination at reducing bacterial viability. When takenalone, no effect would be expected from 3.13 μg/L penicillin with 2.5%MSM, however an 8% reduction in viability is detected (see Table 45-23).These effects are more pronounced with the combination of 6.25 μg/Lpenicillin with MSM. For example, 5% MSM with 6.25 μg/L penicillin wouldbe expected to yield a 96% viable bacterial population (see Table45-24). However, data indicate that viability was reduced to about 17%,nearly an 80% reduction from expected results. Synergy was not detectedwhen 12.5 μg/L penicillin was used, due to the efficacy of thatconcentration of penicillin alone.

TABLE 45-23 S pyogenes Viability After Exposure to 3.13 μg/L ofPenicillin with Various Concentrations of MSM Penicillin (μg/L) MSM (%)S. pyogenes viability (%) 3.13 0 100 3.13 2.5 92.89* 3.13 5.0 78.31*3.13 8.0 9.91*

TABLE 45-24 S pyogenes Viability After Exposure to 6.25 μg/L ofPenicillin with Various Concentrations of MSM Penicillin (μg/L) MSM (%)S. pyogenes viability (%) 6.25 0 100 6.25 2.5 90.11* 6.25 5.0 17.42*6.25 8.0 10.77*

TABLE 45-25 S pyogenes Viability After Exposure to 12.5 μg/L ofPenicillin with Various Concentrations of MSM Penicillin (μg/L) MSM (%)S. pyogenes viability (%) 12.5 0 13.16 12.5 2.5 16.33 12.5 5.0 12.8512.5 8.0 16.02

As with S. pneumonia, combinations of various concentrations of DMSO,MSM, and penicillin were evaluated for their effects on bacterialviability and possible synergistic activity as compared to MSM withpenicillin or DMSO with penicillin Results are shown in Table 45-26.Synergy as compared to DMSO and penicillin is indicated by an “*” whilesynergy as compared to MSM and penicillin is indicated by an “ψ”. As canbe seen by the data in Table 45-26, substantial synergy was detectedacross the various concentrations of compounds. Most combinations ofDMSO and MSM exhibited a dose-response curve based on the concentrationof penicillin used. Based on the efficacy of 12.5 μg/L alone, it is notunexpected that combinations of this concentration of penicillin withDMSO and MSM should be more effective. Of interest, the previouslyineffective concentrations of penicillin are rendered effective in adose dependent manner by combination with DMSO and MSM. For example,2.5% DMSO with 5% MSM and 3.125 μg/L penicillin would be expected toreduce bacterial viability to between 100% and 96% (when compared toDMSO+penicillin and MSM+penicillin, respectively). However, thecombination of all three reduced bacterial viability to about 19%. Theexpected results are similar for combinations with 6.25 μg/L penicillin,but the actual combination reduced bacterial viability even further, toabout 13%. Increasing concentrations of the various compounds does notresult in larger reductions in bacterial viability. For example, thecombination of 8% DMSO with 2.5% MSM and 3.125 μg/L penicillin appearsto be more effective than 8% DMSO with 2.5% MSM and 12.5 μg/L penicillin

TABLE 45-26 S. pneumonia Viability After Exposure to Variouscombinations of DMSO, MSM, and Penicillin DMSO (%) MSM (%) Penicillin(μg/L) S. pyogenes viability (%) 2.5 2.5 3.125 91.74*^(,ψ) 2.5 2.5 6.2560.55*^(,ψ) 2.5 2.5 12.5 8.08*^(,ψ) 2.5 5 3.125 18.72*^(,ψ) 2.5 5 6.2513.38*^(,ψ) 2.5 5 12.5 9.41* 2.5 10 3.125 16.05*^(,ψ) 2.5 10 6.2511.78*^(,ψ) 2.5 10 12.5 11.77* 5 2.5 3.125 14.60*^(,ψ) 5 2.5 6.2510.44*^(,ψ) 5 2.5 12.5 9.55*^(,ψ) 8 2.5 3.125 9.55*^(,ψ) 8 2.5 6.2510.28^(ψ) 8 2.5 12.5 15.55^(ψ)

These studies indicate that at certain concentrations MSM, DMSO or acombination thereof can inhibit Streptococcus pyogenes and StreptococcusPneumonia supporting a possible use of such substances to prevent orinhibit Streptococcus pyogenes and Streptococcus Pneumonia growth.

Example 22 Probiotic Growth in Media Supplemented with MSM

This example describes probiotic growth in media supplemented with MSM.

Lactobacillus acidophilus, Bifidobacterium bifidum, Lactobacillusdelbrueckii, and Bacillus coagulans, growth media were supplemented withMSM at 0, 0.125, 0.25, 0.5, 1.0, 2.5, and 5%. A single 5% MSM stock ofMRS broth was prepared and used to make each media composition. Mediafor the lactobacillus organisms was prepared adding the appropriateamount of MSM to 99 mL of MRS broth. For Bifidobacterium bifidum, 99 mLof MRS broth was prepared with the respective MSM concentrations and0.05% L-cysteine. For Bacillus coagulans, 99 mL of tryptic soy broth wassupplemented with the appropriate amount of MSM.

These media solutions were inoculated with each probiotic organism andincubated at 35° C.±0.5° C. in CO₂ for a total of 72 hours for allsolutions, except Bifidobacterium bifidum, which was grown in anaerobicconditions. Samples of each media were collected at 0, 8, 16, 24, 32,40, 48, 56, 64, and 72 hours. Lactobacillus samples were plated on MRSagar, Bifidobacterium bifidum samples were plated on MRS+L-cysteineagar, and Bacillus coagulans samples were plated on tryptic soy agar.Plates were incubated at 35° C.±0.5° C. in CO₂ for a total of 72 hoursfor all solutions, except Bacillus coagulans, which was grown for 48hours. Plates were then counted. Negative controls (stock media andplating controls) were free of microbial growth. Data are presented inCfu/mL. Results from these studies are presented in the below Tables.

TABLE 46 Growth of Lactobacillus acidophilus in Media Fortified with MSM0.125% 0.2% 0.50% 1% 2.5% 5% Time 0% MSM MSM MSM MSM MSM MSM MSM 0 1.481.37 1.37 1.30 1.48 1.48 1.52 8 1.48 2.19 1.43 2.01 2.25 2.20 1.37 164.87 4.83 5.74 3.82 3.79 4.24 2.69 24 6.97 7.14 8.19 6.47 6.77 5.78 5.3632 9.47 9.15 9.85 9.15 9.05 9.16 8.91 40 7.08 9.53 9.50 9.58 9.49 9.359.25 48 7.27 9.59 9.32 10.45 10.98 10.16 10.88 56 7.20 10.16 9.26 9.3310.90 11.10 10.01 64 7.29 9.40 9.37 9.53 10.34 11.58 8.95 72 7.19 8.568.36 8.57 8.68 8.44 6.66

TABLE 47 Growth of Lactobacillus bulgaricus in Media Fortified with MSM0% 0.125% 0.2% 0.50% 1% 2.5% 5% Time MSM MSM MSM MSM MSM MSM MSM 0 2.222.29 2.25 2.23 2.29 2.26 2.19 8 3.56 4.15 4.08 4.42 4.83 4.57 3.28 168.09 8.22 8.34 8.28 8.36 8.09 7.39 24 8.71 9.04 9.10 9.05 9.03 9.01 8.5032 9.29 8.55 9.60 9.54 9.31 9.15 9.29 40 9.32 9.29 9.11 9.40 9.34 9.279.37 48 10.81 10.94 11.07 10.82 11.23 11.37 10.92 56 7.69 8.00 8.79 9.148.11 8.23 10.07 64 8.78 8.59 8.79 8.80 6.50 8.75 10.96 72 6.56 6.74 6.726.72 6.45 6.51 8.62

TABLE 48 Growth of Bacillus coagulans in Media Fortified with MSM 0%0.125% 0.2% 0.50% 1% 2.5% 5% Time MSM MSM MSM MSM MSM MSM MSM 0 1.431.52 1.67 1.48 1.30 1.43 1.56 8 5.05 4.81 4.94 4.42 4.98 5.13 4.61 166.75 6.95 7.19 6.94 7.29 7.05 7.56 24 10.34 9.87 10.34 10.29 10.30 10.2210.48 32 10.70 11.06 11.25 11.05 11.42 11.70 11.55 40 10.70 11.72 11.3410.25 11.02 10.55 10.85 48 11.07 11.56 9.94 10.40 10.38 10.88 10.22 5611.35 9.60 11.45 10.76 10.86 10.86 11.1 64 11.01 12.13 11.37 10.45 10.4010.97 10.75 72 10.92 10.14 10.94 10.86 10.70 11.05 11.81

TABLE 49 Growth of Bifidobacteruim bifidum in Media Fortified with MSM0% 0.125% 0.2% 0.50% 1% 2.5% 5% Time MSM MSM MSM MSM MSM MSM MSM 0 1.671.64 1.82 1.85 1.48 1.64 1.00 8 2.30 1.73 2.08 1.99 1.70 1.60 2.29 165.33 6.55 5.22 6.53 6.81 6.21 6.71 24 5.86 2.70 6.15 3.14 2.75 2.52 5.7232 8.80 3.37 5.03 3.52 3.37 3.37 10.32 40 9.71 4.19 8.14 4.62 3.48 3.5212.02 48 10.60 6.41 8.55 4.51 3.90 3.95 10.54 56 10.42 9.97 9.00 6.058.32 8.35 10.92 64 10.65 11.34 10.19 9.55 8.02 8.30 12.04 72 11.21 10.009.10 7.52 9.52 10.12 12.43

These studies indicate that MSM can enhance the growth of probioticorganisms depending upon the concentration of MSM employed.

Example 23 Effect of MSM on H1N1 and Herpes Simplex Virus

This example shows the ability of MSM to enhance or reduce infectivityof Swine-like H1N1 Influenza A virus strain A/California/04/2009 (CDC ID#2009712047), Rhinovirus type 14 (ATCC #VR-284), and Herpes SimplexVirus type 1 (ATCC #VR-260). The study was performed as a Pre-treatmenttest of eight concentrations of MSM. Virus yield reduction/enhancementtest and subsequent virus titration was conducted in three replicates.The inhibitory concentrations of MSM (IC₅₀ or IC₉₀—the concentration atwhich growth or activity is inhibited by 50% or 90%) were alsodetermined in this study.

Cytotoxicity of MSM was determined prior to the test. Eightconcentrations of MSM (16%, 14%, 12%. 10%, 8.0%, 6.0%. E0%, and 0.5%)were tested on MDCK cells (ATCC # CCL-34). MSM concentrations 16%through 8% were toxic for MDCK cells and completely destroyed cellmonolayers. Concentrations 6% through 0.5% produced no visible cytotoxiceffect. TC₅₀ (concentration at which the compound, alone, kills 50% ofuninfected cells) was determined to be approximately 7%. Therefore, thisconcentration was the first lowest non-cytotoxic dilution used intesting.

A total of eight MSM concentrations were included in testing: 7%(˜74.365 mM); 6% (˜63.742 mM); 5% (˜53.118 mM); 4% (˜42.494 mM); 3%(˜31.871 mM); 2% (˜21.247 mM); 1% (˜10.624 mM); and 0.5% (˜5.312 mM). Adetailed description of the material and methods are provided below.

Host Cells. Madin Darby Canis Kidney (MDCK [ATCC#CCL-34]) cells, MRC-5(Human lung fibroblasts; [ATCC #CCL-171]) cells, and Vero (African greenmonkey kidney [ATCC#CCL-81]) cells were maintained as monolayers indisposable cell culture labware and were used for the Pre-TreatmentAntiviral Test of Swine-like H1N1 Influenza A virus strainA/California/04/2009, Rhinovirus type 14 (ATCC #VR-284), and HSV-1 (ATCC#VR-260), respectively. Prior to testing, host cell cultures were seededonto the appropriate cell culture plates. Cell monolayers were 80 to 90%confluent and less than 48 hours old before inoculation with the virus.The growth medium (GM) and maintenance medium (MM) was 1×EMEM and/orAdvanced MEM with appropriate supplements.

Determination of test product cytotoxicity. The highest non-cytotoxicconcentration of the test product was determined prior to the test. MDCKcell culture was washed with Phosphate Buffered Saline (PBS) andincubated with the following dilutions of a product: 16%, 14%, 12%, 10%,8.0%, 6.0%, 1.0%, and 0.5%. Incubation was 1 hour at 37°±2° C. in a CO₂incubator. After incubation, the treated cells were overlaid with MM.The plates were incubated in a CO₂ incubator for 3 days at 37°±2° C.Toxicity was monitored using an Inverted Compound Microscope. Acytotoxicity test performed as outlined in the Study Protocol, showedthat the product concentrations 16% through 8% were toxic for MDCK cellsand completely destroyed cell monolayers. Product concentrations 6%through 0.5% produced no visible cytotoxic effect. TC₅₀ (concentrationat which the compound, alone, kills 50% of uninfected cells) wasdetermined to be approximately 7%.

A. Pre-Treatment Test, Test Product stock solution was prepared asfollows: 35.0 gram of product was diluted in 100 mL PBS and heated at40° C. until dissolved. The 35% solution was kept at 40° C. until higherdilutions were prepared (see Project Notes [Form No. 95-G-001] inAddendum VI of this Final Report). MDCK, MRC-5 and Vero cell cultureswere washed with PBS and incubated with the following product dilutions:7%, 6%, 5%, 4%, 3%, 2%, 1%, and 0.5%. Incubation was 1 hour at 37°±2° C.in a CO₂ incubator. After incubation had been completed approximately300-1000 IU (infectious units) of each of the test viruses were added tothe appropriate treated cells. The testing was performed in threereplicates. The plates were incubated in a CO₂ incubator for 6 days atthe temperature appropriate for each virus. CPE was monitored using anInverted Compound Microscope. All data resulting from the test areincluded in Addendum IV of this Final Report (Forms No.: 95-G-001,91-L-002, and 07-L-002).

B. Toxicity Control for Pre-Treatment Test. MDCK, MRC-5 and Vero cellcultures were washed with PBS and incubated with the product dilutions7% through 0.5%. Incubation was 1 hour at 37°±2° C. in a CO₂ incubator.After incubation, the treated cells were overlaid with MM. The plateswere incubated in a CO₂ incubator for 6 days at the temperatureappropriate for each virus. Toxicity was monitored using an InvertedCompound Microscope. The results of the cytotoxicity tests are presentedin Table 50.

C. Virus Control. MDCK, MRC-5 and Vero cell cultures were washed withPBS and incubated with MM. Incubation was 1 hour at 37°±2° C. in a CO₂incubator. After incubation had been completed approximately 300-1000 IU(infectious units) of each of the test viruses were added to the cells.Three replicates of Virus Control were performed. The plates wereincubated in a CO₂ incubator for 6 days at the temperature appropriatefor each virus. CPE was monitored using an Inverted Compound Microscope.

D. Negative Control. Intact cell culture monolayers served as thenegative control. The GM was replaced by MM in all Negative controlwells.

E. Determination of the reduction and/or enhancement of virus yield.After Virus Control had reached the maximum cytopathic effect (completedestruction of the monolayer), samples from the test wells and viruscontrol wells were taken for titration. Ten-fold dilutions were made inMM and plated onto susceptible cells in four replicates. The results ofthe virus yield reduction/enhancement tests are presented in the Tables51 through 91.

Data Analysis. Virus population titer in cell cultures were expressed as−log₁₀ of the 50% titration end point for infectivity. To calculate theviral titer, a 50% tissue culture infectious dose (TCID₅₀)calculation—the Quantal test (Spearman-Kärber Method)—was applied.

log TCID₅₀=1−d(s−0.5)

-   -   Where:        -   l=−log₁₀ of the lowest dilution;        -   d=difference between dilution steps;        -   s=sum of proportions of positive wells.    -   1.1 The highest compound concentration that produces a cytotoxic        effect was determined as 50% of the toxic compound concentration        (TC₅₀).    -   1.2 The percent reduction was calculated as follows:

${\% \mspace{14mu} {Reduction}} = {\left\lbrack {1 - \frac{{TCID}_{50}\mspace{14mu} {test}}{{TCID}_{50}\mspace{14mu} {virus}\mspace{14mu} {control}}} \right\rbrack \times 100}$

-   -   1.3 TCID₅₀ of virus population recovered from the test and virus        control was used to calculate reduction or enhancement of virus        infectivity. IC₅₀ was determined using GraphPad Prism 5, Inc.        software. IC₉₀ was determined experimentally when present.        Test Acceptance Criteria. A valid test requires that: 1) cells        in the Negative control wells are viable and attached to the        bottom of the well; 2) the medium be free of contamination in        all wells of the plate; and 3) Virus Control shows the presence        of virus-specific CPE.

Reductions of virus population were observed for all three test viruses.MSM at 7% concentration produced following average reductions: 1.16log₁₀ reduction (93.08% reduction) of Swine-like H1N1 Influenza A virus;2.50 log₁₀ reduction (99.68% reduction) of Herpes Simplex Virus type 1(HSV-1); 1.25 log₁₀ reduction (94.38% reduction) of Rhinovirus type 14.MSM at 6% concentration produced following average reductions: 1.00log₁₀ reduction (90.00% reduction) of Swine-like H1N1 Influenza A virus;1.00 log₁₀ reduction (90.00% reduction) of HSV-1; 0.67 log₁₀ reduction(78.62% reduction) of Rhinovirus type 14.

MSM at 5% concentration produced following average reductions: 0.41log₁₀ reduction (61.10% reduction) of Swine-like H1N1 Influenza A virus;1.34 log₁₀ reduction (95.43% reduction) of HSV-1; 0.09 log₁₀ reduction(18.72% reduction) of Rhinovirus type 14. MSM at 4% concentrationproduced following average reductions: 0.16 log₁₀ reduction (30.82%reduction) of Swine-like H1N1 Influenza A virus; 1.59 log₁₀ reduction(97.43% reduction) of HSV-1; 0.28 log₁₀ reduction (47.52% reduction) ofRhinovirus type 14. MSM at 3% concentration produced following averagereductions: 0.00 log₁₀ reduction (00.00% reduction) of Swine-like H1N1Influenza A virus; 1.00 log₁₀ reduction (90.00% reduction) of HSV-1;0.11 log₁₀ reduction (22.38% reduction) of Rhinovirus type 14. MSM at 2%concentration produced following average reductions: 0.41 log₁₀reduction (61.10% reduction) of Swine-like H1N1 Influenza A virus; 0.84log₁₀ reduction (85.55% reduction) of HSV-1; 0.42 log₁₀ reduction(61.98% reduction) of Rhinovirus type 14. MSM at 1% concentrationproduced following average reductions: 0.25 log₁₀ reduction (43.77%reduction) of Swine-like H1N1 Influenza A virus; 0.67 log₁₀ reduction(78.62% reduction) of HSV-1; 0.14 log₁₀ reduction (27.56% reduction) ofRhinovirus type 14. MSM at 0.5% concentration produced following averagereductions: 0.66 log₁₀ reduction (78.12% reduction) of Swine-like H1N1Influenza A virus; 0.25 log₁₀ reduction (43.77% reduction) of HSV-1;0.40 log₁₀ reduction (60.19% reduction) of Rhinovirus type 14.

Enhancement/stimulation of virus infectivity was observed for Swine-likeH1N1 Influenza A virus treated with 3% MSM. The average enhancement ofvirus population was 0.17 log₁₀ (32.39%). A total of three MSMconcentrations enhanced infectivity of Rhinovirus type 14. MSM at 5%concentration produced an average of 0.053 log₁₀ enhancement (11.49%).Three percent MSM produced an average of 0.11 log₁₀ enhancement(22.38%); and 1% MSM produced an average of 0.11 log₁₀ enhancement(22.38%). All of the virus infectivity enhancements/stimulationsdetermined in this study were situated within the range of normalvariation for virus population and were not significant. An Inhibitoryconcentration of MSM at which growth or activity is inhibited by 50%(IC₅₀) was computed using nonlinear regression Dose-Response (GraphPadPrism 5, software). The best-fit MSM IC₅₀ and IC₅₀ values with 95%confidence intervals were calculated for each test virus. For Swine-likeH1N1 Influenza A virus, the best-fit value of MSM IC₅₀ was 5.114 mM.IC₅₀ with 95% confidence interval was varying from 0.008038 mM to 3253mM. For HSV-1, the best-fit value of MSM IC₅₀ was determined to be 10.13mM with an IC₅₀ with 95% confidence interval varying from 7.144 mM to14.37 mM. For Rhinovirus type 14, the best fit value of MSM IC₅₀ was38.16 mM. IC_(so) with 95% confidence interval was in the range of 13.07mM to 111.4 mM. IC₉₀ (1.0 log₁₀ reduction) were determinedexperimentally for HSV-1 and Swine-like Influenza A H1N1. However, dueto interception of multiple MSM concentrations with the 90% reductionaxis, IC₉₀ experimental values cannot be considered precise.

MSM tested at eight different concentrations against HSV-1, Swine-likeInfluenza A H1N1 and Rhinovirus produced U-shaped dose-response curves.For instance: 4% MSM (1.00 log₁₀ reduction) was more effective againstHSV-1 than 6% MSM (1.59 log₁₀ reduction); 0.5% MSM (0.66 log₁₀reduction) was more or equally effective against Swine-like Influenza AH1N1 than 5% MSM (0.41 log₁₀ reduction); concentrations 4% through 0.5%were more or equally effective against Rhinovirus than 5% MSM. It ispossible, if confirmed in further research, that U-shaped MSM effectsrepresent a stable event.

This study indicates MSM can be used as an antiviral product.Non-cytotoxic concentrations of 7% and 6% reduced populations ofenveloped viruses such as HSV-1 and Swine-like Influenza A H1N1 by morethan 1.0 log₁₀. Tables 50 through 91 include the results for theaforementioned studies.

Table 50 presents Cytotoxicity Test for eight product concentrationsperformed in parallel with Pre-Treatment test using MDCK, MRC-5, andVero cell cultures.

TABLE 50 Test Product: Methylsulfonylmethane, lot# 0902951 Cell CultureTest Product Cytotoxicity Designation 7% 6% 5% 4% 3% 2% 1% 0.5% Vero 0000 00 00 00 00 00 00 MDCK ++ ++ 00 00 00 00 00 00 MRC-5 ++ 00 00 00 0000 00 00 + = CPE Present 0 = CPE not detectedTables 2 through 9 present the Virus Control infectivity (TCID₅₀), theaverage infectivity (TCID₅₀), and the log₁₀ and percent reductionsobserved in Pre-treatment test of the Test Product,Methylsulfonylmethane (Lot Number 0902951), and Swine-like H1N1Influenza A virus strain A/California/04/2009 (CDC ID #2009712047).

TABLE 51 Reduction of Infectivity Test Product: Methylsulfonylmethane,7% (lot# 0902951) Virus: Swine-like Influenza A H1N1 strainA/California/04/2009 CDC ID # 2009712047 Host Cell Line: MDCK Host CellLine ATCC # CCL-34 Dilutions Virus Control Test Product (−log₁₀) Rep. 1Rep. 2 Rep. 3 Rep. 1 Rep. 2 Rep. 3 Cell Control 0000 −2 NT NT NT ++++++++ ++++ −3 ++++ ++++ ++++ ++++ ++++ ++++ −4 ++++ ++++ ++++ 00+0 00000+00 −5 00+0 +000 +0+0 0000 0000 0000 −6 0000 0000 0000 0000 0000 0000−7 0000 0000 0000 0000 0000 0000 TCID₅₀ 4.75log₁₀ 4.75log₁₀ 5.00log₁₀3.75log₁₀ 3.50log₁₀ 3.75log₁₀ Average 4.83log₁₀ 3.67log₁₀ TCID₅₀ Log1.08log₁₀ 1.33log₁₀ 1.08log₁₀ Reduction* Average 1.16log₁₀ Log ReductionPercent 91.68% 95.32% 91.68% Reduction Average 93.08% PercentReduction** + = CPE Present 0 = CPE not detected NT = Not Tested Rep =Replicate *Log Reduction = Average TCID₅₀ of Virus Control − TCID₅₀ ofthe Test Replicate **Average % Reduction (calculated from average log₁₀reduction) = 100 − (1/TCID₅₀ Reduction) * 100

TABLE 52 Reduction of Infectivity Test Product: Methylsulfonylmethane,6% (lot# 0902951) Virus: Swine-like Influenza A H1N1 strainA/California/04/2009 CDC ID # 2009712047 Host Cell Line: MDCK Host CellLine ATCC # CCL-34 Dilutions Virus Control Test Product Cell (−log₁₀)Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2 Rep. 3 Control 0000 −2 NT NT NT ++++++++ ++++ −3 ++++ ++++ ++++ ++++ ++++ ++++ −4 ++++ ++++ ++++ 0+00 000++00+ −5 00+0 +000 +0+0 0000 0000 0000 −6 0000 0000 0000 0000 0000 0000−7 0000 0000 0000 0000 0000 0000 TCID₅₀ 4.75log₁₀ 4.75 log ₁₀ 5.00 log₁₀ 3.75log₁₀ 3.75log₁₀ 4.00log₁₀ Average TCID₅₀ 4.83log₁₀ 3.67log₁₀ LogReduction* 1.08log₁₀ 1.08log₁₀ 0.83log₁₀ Average Log 1.00log₁₀ ReductionPercent Reduction 91.68% 91.68% 85.21% Average Percent 90.00%Reduction** + = CPE Present 0 = CPE not detected NT = Not Tested Rep =Replicate *Log Reduction = Average TCID₅₀ of Virus Control − TCID₅₀ ofthe Test Replicate **Average % Reduction (calculated from average log₁₀reduction) = 100 − (1/TCID₅₀ Reduction) * 100

TABLE 53 Reduction of Infectivity Test Product: Methylsulfonylmethane,5% (lot# 0902951) Virus: Swine-like Influenza A H1N1 strainA/California/04/2009 CDC ID # 2009712047 Host Cell Line: MDCK Host CellLine ATCC # CCL-34 Dilutions Virus Control Test Product (−log₁₀) Rep. 1Rep. 2 Rep. 3 Rep. 1 Rep. 2 Rep. 3 Cell Control 0000 −2 NT NT NT ++++++++ ++++ −3 ++++ ++++ ++++ ++++ ++++ ++++ −4 ++++ ++++ ++++ ++++ 0++++++0 −5 00+0 +000 +0+0 0000 +000 0000 −6 0000 0000 0000 0000 0000 0000−7 0000 0000 0000 0000 0000 0000 TCID₅₀ 4.75log₁₀ 4.75log₁₀ 5.00log₁₀4.50log₁₀ 4.50log₁₀ 4.25log₁₀ Average 4.83log₁₀ 4.42log₁₀ TCID₅₀ Log0.33log₁₀ 0.33log₁₀ 0.58log₁₀ Reduction* Average 0.41log₁₀ Log ReductionPercent 53.23% 53.23% 73.70% Reduction Average 61.10% PercentReduction** + = CPE Present 0 = CPE not detected NT = Not Tested Rep =Replicate *Log Reduction = Average TCID₅₀ of Virus Control − TCID₅₀ ofthe Test Replicate **Average % Reduction (calculated from average log₁₀reduction) = 100 − (1/TCID₅₀ Reduction) * 100

TABLE 54 Reduction of Infectivity Test Product: Methylsulfonylmethane,4% (lot# 0902951) Virus: Swine-like Influenza A H1N1 strainA/California/04/2009 CDC ID # 2009712047 Host Cell Line: MDCK Host CellLine ATCC # CCL-34 Dilutions Virus Control Test Product (−log₁₀) Rep. 1Rep. 2 Rep. 3 Rep. 1 Rep. 2 Rep. 3 Cell Control 0000 −2 NT NT NT ++++++++ ++++ −3 ++++ ++++ ++++ ++++ ++++ ++++ −4 ++++ ++++ ++++ ++++ 0+++++++ −5 00+0 +000 +0+0 00+0 000+ 000+ −6 0000 0000 0000 0000 0000 0000−7 0000 0000 0000 0000 0000 0000 TCID₅₀ 4.75log₁₀ 4.75log₁₀ 5.00log₁₀4.75log₁₀ 4.50log₁₀ 4.75log₁₀ Average 4.83log₁₀ 4.67log₁₀ TCID₅₀ Log0.08log₁₀ 0.33log₁₀ 0.08log₁₀ Reduction* Average 0.16log₁₀ Log ReductionPercent 16.82% 53.23% 16.82% Reduction Average 30.82% PercentReduction** + = CPE Present 0 = CPE not detected NT = Not Tested Rep =Replicate *Log Reduction = Average TCID₅₀ of Virus Control − TCID₅₀ ofthe Test Replicate **Average % Reduction (calculated from average log₁₀reduction) = 100 − (1/TCID₅₀ Reduction) * 100

TABLE 55 Reduction of Infectivity Test Product: Methylsulfonylmethane,3% (lot# 0902951) Virus: Swine-like Influenza A H1N1 strainA/California/04/2009 CDC ID # 2009712047 Host Cell Line: MDCK Host CellLine ATCC # CCL-34 Dilutions Virus Control Test Product (−log₁₀) Rep. 1Rep. 2 Rep. 3 Rep. 1 Rep. 2 Rep. 3 Cell Control 0000 −2 NT NT NT ++++++++ ++++ −3 ++++ ++++ ++++ ++++ ++++ ++++ −4 ++++ ++++ ++++ ++++ ++++++++ −5 00+0 +000 +0+0 00++ +00+ 0++0 −6 0000 0000 0000 0000 0000 0000−7 0000 0000 0000 0000 0000 0000 TCID₅₀ 4.75log₁₀ 4.75log₁₀ 5.00log₁₀5.00log₁₀ 5.00log₁₀ 5.00log₁₀ Average 4.83log₁₀ 5.00log₁₀ TCID₅₀ Log0.00log₁₀ 0.00log₁₀ 0.00log₁₀ Reduction Average 0.00log₁₀ Log ReductionPercent 00.00% 00.00% 00.00% Reduction Average 00.00% PercentReduction + = CPE Present 0 = CPE not detected NT = Not Tested Rep =Replicate *Log Reduction = Average TCID₅₀ of Virus Control − TCID₅₀ ofthe Test Replicate **Average % Reduction (calculated from average log₁₀reduction) = 100 − (1/TCID₅₀ Reduction) * 100

TABLE 56 Reduction of Infectivity Test Product: Methylsulfonylmethane,2% (lot# 0902951) Virus: Swine-like Influenza A H1N1 strainA/California/04/2009 CDC ID # 2009712047 Host Cell Line: MDCK Host CellLine ATCC # CCL-34 Dilutions Virus Control Test Product (−log₁₀) Rep. 1Rep. 2 Rep. 3 Rep. 1 Rep. 2 Rep. 3 Cell Control 0000 −2 NT NT NT ++++++++ ++++ −3 ++++ ++++ ++++ ++++ ++++ ++++ −4 ++++ ++++ ++++ ++++ +++++000 −5 00+0 +000 +0+0 0000 000+ 0+00 −6 0000 0000 0000 0000 0000 0000−7 0000 0000 0000 0000 0000 0000 TCID₅₀ 4.75log₁₀ 4.75log₁₀ 5.00log₁₀4.50log₁₀ 4.75log₁₀ 4.00log₁₀ Average 4.83log₁₀ 4.42log₁₀ TCID₅₀ Log0.33log₁₀ 0.08log₁₀ 0.83log₁₀ Reduction* Average 0.41log₁₀ Log ReductionPercent 53.23% 16.82% 85.21% Reduction Average 61.10% PercentReduction** + = CPE Present 0 = CPE not detected NT = Not Tested Rep =Replicate *Log Reduction = Average TCID₅₀ of Virus Control − TCID₅₀ ofthe Test Replicate **Average % Reduction (calculated from average log₁₀reduction) = 100 − (1/TCID₅₀ Reduction) * 100

TABLE 57 Reduction of Infectivity Test Product: Methylsulfonylmethane,1% (lot# 0902951) Virus: Swine-like Influenza A H1N1 strainA/California/04/2009 CDC ID # 2009712047 Host Cell Line: MDCK Host CellLine ATCC # CCL-34 Dilutions Virus Control Test Product (−log₁₀) Rep. 1Rep. 2 Rep. 3 Rep. 1 Rep. 2 Rep. 3 Cell Control 0000 −2 NT NT NT ++++++++ ++++ −3 ++++ ++++ ++++ ++++ ++++ ++++ −4 ++++ ++++ ++++ ++0+ ++++++++ −5 00+0 +000 +0+0 00+0 000+ 0000 −6 0000 0000 0000 0000 0000 0000−7 0000 0000 0000 0000 0000 0000 TCID₅₀ 4.75log₁₀ 4.75log₁₀ 5.00log₁₀4.50log₁₀ 4.75log₁₀ 4.50log₁₀ Average 4.83log₁₀ 4.58log₁₀ TCID₅₀ Log0.33log₁₀ 0.08log₁₀ 0.33log₁₀ Reduction* Average 0.25log₁₀ Log ReductionPercent 53.23% 16.82% 53.23% Reduction Average 43.77% PercentReduction** + = CPE Present 0 = CPE not detected NT = Not Tested Rep =Replicate *Log Reduction = Average TCID₅₀ of Virus Control − TCID₅₀ ofthe Test Replicate **Average % Reduction (calculated from average log₁₀reduction) = 100 − (1/TCID₅₀ Reduction) * 100

TABLE 58 Reduction of Infectivity Test Product: Methylsulfonylmethane,0.5% (lot# 0902951) Virus: Swine-like Influenza A H1N1 strainA/California/04/2009 CDC ID # 2009712047 Host Cell Line: MDCK Host CellLineATCC # CCL-34 Dilutions Virus Control Test Product (−log₁₀) Rep. 1Rep. 2 Rep. 3 Rep. 1 Rep. 2 Rep. 3 Cell Control 0000 −2 NT NT NT ++++++++ ++++ −3 ++++ ++++ ++++ ++++ ++++ ++++ −4 ++++ ++++ ++++ +000 +++++++0 −5 00+0 +000 +0+0 0000 0000 0000 −6 0000 0000 0000 0000 0000 0000−7 0000 0000 0000 0000 0000 0000 TCID₅₀ 4.75log₁₀ 4.75log₁₀ 5.00log₁₀3.75log₁₀ 4.50log₁₀ 4.25log₁₀ Average 4.83log₁₀ 4.17log₁₀ TCID₅₀ Log1.08log₁₀ 0.33log₁₀ 0.58log₁₀ Reduction* Average 0.66log₁₀ Log ReductionPercent 91.68% 53.23% 73.70% Reduction Average 78.12% PercentReduction** + = CPE Present 0 = CPE not detected NT = Not Tested Rep =Replicate *Log Reduction = Average TCID₅₀ of Virus Control − TCID₅₀ ofthe Test Replicate **Average % Reduction (calculated from average log₁₀reduction) = 100 − (1/TCID₅₀ Reduction) * 100

Tables 59 through 67 present the Virus Control infectivity (TCID₅₀), theaverage infectivity (TCID₅₀), and the log₁₀ and percent reductionsobserved in Pre-treatment test of the Test Product,Methylsulfonylmethane (Lot Number 0902951), and Herpes Simplex Virustype I (ATCC # VR-260).

TABLE 59 Reduction of Infectivity Test Product: Methylsulfonylmethane,7% (lot# 0902951) Virus: Herpes Simplex Virus strain HF ATCC # VR-260Host Cell Line: Vero Host Cell Line ATCC # CCL-81 Dilutions VirusControl Test Product (−log₁₀) Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2 Rep. 3Cell Control 0000 −1 NT NT NT ++++ ++++ ++++ −2 NT NT NT ++++ ++++ ++++−3 NT NT NT ++++ +000 0+0+ −4 ++++ ++++ ++++ 0000 00+0 0000 −5 ++++ +++0++++ 0000 0000 0000 −6 0000 00+0 +0+0 0000 0000 0000 −7 0000 0000 0000NT NT NT −8 0000 0000 0000 NT NT NT TCID₅₀ 5.50log₁₀ 5.50log₁₀ 6.00log₁₀3.50log₁₀ 3.00log₁₀ 3.00log₁₀ Average 5.67log₁₀ 3.17log₁₀ TCID₅₀ Log2.17log₁₀ 2.67log₁₀ 2.67log₁₀ Reduction* Average 2.50log₁₀ Log ReductionPercent 99.32% 99.79% 99.79% Reduction Average 99.68% PercentReduction** + = CPE Present 0 = CPE not detected NT = Not Tested Rep =Replicate *Log Reduction = Average TCID₅₀ of Virus Control − TCID₅₀ ofthe Test Replicate **Average % Reduction (calculated from average log₁₀reduction) = 100 − (1/TCID₅₀ Reduction) * 100

TABLE 60 Reduction of Infectivity Test Product: Methylsulfonylmethane,6% (lot# 0902951) Virus: Herpes Simplex Virus strain HF ATCC # VR-260Host Cell Line: Vero Host Cell Line ATCC # CCL-81 Dilutions VirusControl Test Product Cell (−log₁₀) Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2Rep. 3 Control 0000 −3 NT NT NT ++++ ++++ ++++ −4 ++++ ++++ ++++ ++++++++ +++0 −5 ++++ +++0 ++++ 000+ 0+00 +000 −6 0000 00+0 +0+0 0000 00000000 −7 0000 0000 0000 0000 0000 0000 −8 0000 0000 0000 0000 0000 0000TCID₅₀ 5.50log₁₀ 5.50log₁₀ 6.00log₁₀ 4.75log₁₀ 4.75log₁₀ 4.50log₁₀Average 5.67log₁₀ 4.67log₁₀ TCID₅₀ Log 0.92log₁₀ 0.92log₁₀ 1.17log₁₀Reduction* Average Log 1.00log₁₀ Reduction Percent 87.98% 87.98% 93.24%Reduction Average 90.00% Percent Reduction** + = CPE Present 0 = CPE notdetected NT = Not Tested Rep = Replicate *Log Reduction = Average TCID₅₀of Virus Control − TCID₅₀ of the Test Replicate **Average % Reduction(calculated from average log₁₀ reduction) = 100 − (1/TCID₅₀ Reduction) *100

TABLE 61 Reduction of Infectivity Test Product: Methylsulfonylmethane,5% (lot# 0902951) Virus: Herpes Simplex Virus strain HF ATCC # VR-260Host Cell Line: Vero Host Cell Line ATCC # CCL-81 Dilutions VirusControl Test Product (−log₁₀) Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2 Rep. 3Cell Control 0000 −3 NT NT NT ++++ ++++ ++++ −4 ++++ ++++ ++++ ++0+ ++++000+ −5 ++++ +++0 ++++ 0+00 0000 0000 −6 0000 00+0 +0+0 000+ 0000 0000−7 0000 0000 0000 0000 0000 0000 −8 0000 0000 0000 0000 0000 0000 TCID₅₀5.50log₁₀ 5.50log₁₀ 6.00log₁₀ 4.75log₁₀ 4.50log₁₀ 3.75log₁₀ Average5.67log₁₀ 4.33log₁₀ TCID₅₀ Log 0.92log₁₀ 1.17log₁₀ 1.92log₁₀ Reduction*Average 1.34log₁₀ Log Reduction Percent 87.98% 93.24% 98.80 ReductionAverage 95.43% Percent Reduction** + = CPE Present 0 = CPE not detectedNT = Not Tested Rep = Replicate *Log Reduction = Average TCID₅₀ of VirusControl − TCID₅₀ of the Test Replicate **Average % Reduction (calculatedfrom average log₁₀ reduction) = 100 − (1/TCID₅₀ Reduction) * 100

TABLE 62 Reduction of Infectivity Test Product: Methylsulfonylmethane,4% (lot# 0902951) Virus: Herpes Simplex Virus strain HF ATCC # VR-260Host Cell Line: Vero Host Cell Line ATCC # CCL-81 Dilutions VirusControl Test Product (−log₁₀) Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2 Rep. 3Cell Control 0000 −3 NT NT NT ++++ ++++ ++++ −4 ++++ ++++ ++++ 00+0 +0+0+0+0 −5 ++++ +++0 ++++ 0000 +00+ 0000 −6 0000 00+0 +0+0 0000 0000 0000−7 0000 0000 0000 0000 0000 0000 −8 0000 0000 0000 0000 0000 0000 TCID₅₀5.50log₁₀ 5.50log₁₀ 6.00log₁₀ 3.75log₁₀ 4.50log₁₀ 4.00log₁₀ Average5.67log₁₀ 4.08log₁₀ TCID₅₀ Log 1.92log₁₀ 1.17log₁₀ 1.67log₁₀ Reduction*Average 1.59log₁₀ Log Reduction Percent 98.80% 93.24% 97.86% ReductionAverage 97.43% Percent Reduction** + = CPE Present 0 = CPE not detectedNT = Not Tested Rep = Replicate *Log Reduction = Average TCID₅₀ of VirusControl − TCID₅₀ of the Test Replicate **Average % Reduction (calculatedfrom average log₁₀ reduction) = 100 − (1/TCID₅₀ Reduction) * 100

TABLE 63 Reduction of Infectivity Test Product: Methylsulfonylmethane,3% (lot# 0902951) Virus: Herpes Simplex Virus strain HF ATCC # VR-260Host Cell Line: Vero Host Cell Line ATCC # CCL-81 Dilutions VirusControl Test Product (−log₁₀) Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2 Rep. 3Cell Control 0000 −3 NT NT NT ++++ ++++ ++++ −4 ++++ ++++ ++++ 00++ ++++++++ −5 ++++ +++0 ++++ 00+0 0000 00++ −6 0000 00+0 +0+0 0000 0000 0000−7 0000 0000 0000 0000 0000 000+ −8 0000 0000 0000 0000 0000 0000 TCID₅₀5.50log₁₀ 5.50log₁₀ 6.00log₁₀ 4.25log₁₀ 4.50log₁₀ 5.25log₁₀ Average5.67log₁₀ 4.67log₁₀ TCID₅₀ Log 1.42log₁₀ 1.17log₁₀ 0.42log₁₀ Reduction*Average 1.00log₁₀ Log Reduction Percent 96.20% 93.24% 61.98% ReductionAverage 90.00% Percent Reduction** + = CPE Present 0 = CPE not detectedNT = Not Tested Rep = Replicate *Log Reduction = Average TCID₅₀ of VirusControl − TCID₅₀ of the Test Replicate **Average % Reduction (calculatedfrom average log₁₀ reduction) = 100 − (1/TCID₅₀ Reduction) * 100

TABLE 64 Reduction of Infectivity Test Product: Methylsulfonylmethane,2% (lot# 0902951) Virus: Herpes Simplex Virus strain HF ATCC # VR-260Host Cell Line: Vero HostCell Line ATCC # CCL-81 Dilutions Virus ControlTest Product Cell (−log₁₀) Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2 Rep. 3Control 0000 −3 NT NT NT ++++ ++++ ++++ −4 ++++ ++++ ++++ ++++ ++++ ++++−5 ++++ +++0 ++++ 0+++ 0000 00+0 −6 0000 00+0 +0+0 0000 0000 0000 −70000 0000 0000 0000 0000 0000 −8 0000 0000 0000 0000 0000 0000 TCID₅₀5.50log₁₀ 5.50log₁₀ 6.00log₁₀ 5.25log₁₀ 4.50log₁₀ 4.75log₁₀ Average5.67log₁₀ 4.83log₁₀ TCID₅₀ Log 0.42log₁₀ 1.17log₁₀ 0.92log₁₀ ReductionAverage 0.84log₁₀ Log Reduction Percent 61.98% 93.24% 87.98% ReductionAverage 85.55% Percent Reduction + = CPE Present 0 = CPE not detected NT= Not Tested Rep = Replicate *Log Reduction = Average TCID₅₀ of VirusControl − TCID₅₀ of the Test Replicate **Average % Reduction (calculatedfrom average log₁₀ reduction) = 100 − (1/TCID₅₀ Reduction) * 100

TABLE 65 Reduction of Infectivity Test Product: Methylsulfonylmethane,1% (lot# 0902951) Virus: Herpes Simplex Virus strain HF ATCC # VR-260Host Cell Line: Vero HostCell Line ATCC # CCL-81 Dilutions Virus ControlTest Product Cell (−log₁₀) Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2 Rep. 3Control 0000 −3 NT NT NT ++++ ++++ ++++ −4 ++++ ++++ ++++ ++++ ++++ ++++−5 ++++ +++0 ++++ 00++ 00++ 00++ −6 0000 00+0 +0+0 0000 0000 0000 −70000 0000 0000 0000 0000 0000 −8 0000 0000 0000 0000 0000 0000 TCID₅₀5.50log₁₀ 5.50log₁₀ 6.00log₁₀ 5.00log₁₀ 5.00log₁₀ 5.00log₁₀ Average5.67log₁₀ 5.00log₁₀ TCID₅₀ Log 0.67log₁₀ 0.67log₁₀ 0.67log₁₀ ReductionAverage 0.67log₁₀ Log Reduction Percent 78.62% 78.62% 78.62% ReductionAverage 78.62% Percent Reduction + = CPE Present 0 = CPE not detected NT= Not Tested Rep = Replicate *Log Reduction = Average TCID₅₀ of VirusControl − TCID₅₀ of the Test Replicate **Average % Reduction (calculatedfrom average log₁₀ reduction) = 100 − (1/TCID₅₀ Reduction) * 100

TABLE 66 Reduction of Infectivity Test Product: Methylsulfonylmethane,0.5% (lot# 0902951) Virus: Herpes Simplex Virus strain HF ATCC # VR-260Host Cell Line: Vero Host Cell Line ATCC # CCL-81 Dilutions VirusControl Test Product Cell (−log₁₀) Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2Rep. 3 Control 0000 −3 NT NT NT ++++ ++++ ++++ −4 ++++ ++++ ++++ ++++++++ ++++ −5 ++++ +++0 ++++ ++++ ++0+ ++0+ −6 0000 00+0 +0+0 0000 0000000+ −7 0000 0000 0000 0000 0000 0000 −8 0000 0000 0000 0000 0000 0000TCID₅₀ 5.50log₁₀ 5.50log₁₀ 6.00log₁₀ 5.50log₁₀ 5.25log₁₀ 5.50log₁₀Average 5.67log₁₀ 5.42log₁₀ TCID₅₀ Log 0.17log₁₀ 0.42log₁₀ 0.17log₁₀Reduction Average 0.25log₁₀ Log Reduction Percent 32.39% 61.98% 32.39%Reduction Average 43.77% Percent Reduction + = CPE Present 0 = CPE notdetected NT = Not Tested Rep = Replicate * - Log Reduction = AverageTCID₅₀ of Virus Control − TCID₅₀ of the Test Replicate ** - Average %Reduction (calculated from average log₁₀ reduction) = 100 − (1/TCID₅₀Reduction) * 100

TABLE 67 Reduction of Infectivity Test Product: Methylsulfonylmethane,7% (lot# 0902951) Virus: Rhinovirus type 14 strain 1059 ATCC # VR-284Host Cell Line: MRC-5 Host Cell Line ATCC # CCL-171 Dilutions VirusControl Test Product Cell (−log₁₀) Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2Rep. 3 Control 0000 −2 NT NT NT ++++ ++++ ++++ −3 ++++ ++++ ++++ ++++++++ ++++ −4 ++++ ++++ ++++ ++0+ ++++ ++++ −5 ++++ ++++ ++++ 0000 00000000 −6 0000 0000 0+0+ 0000 0000 0000 −7 0000 0000 0000 0000 0000 0000TCID₅₀ 5.50log₁₀ 5.50log₁₀ 6.00log₁₀ 4.25log₁₀ 4.50log₁₀ 4.50log₁₀Average 5.67log₁₀ 4.42log₁₀ TCID₅₀ Log 1.42log₁₀ 1.17log₁₀ 1.17log₁₀Reduction Average 1.25log₁₀ Log Reduction Percent 96.20% 93.24% 93.24%Reduction Average 94.38% Percent Reduction + = CPE Present 0 = CPE notdetected NT = Not Tested Rep = Replicate * - Log Reduction = AverageTCID₅₀ of Virus Control − TCID₅₀ of the Test Replicate ** - Average %Reduction (calculated from average log₁₀ reduction) = 100 − (1/TCID₅₀Reduction) * 100

Tables 68 through 74 present the Virus Control infectivity (TCID₅₀), theaverage infectivity (TCID₅₀), and the log₁₀ and percent reductionsobserved in Pre-treatment test of the Test Product,Methylsulfonylmethane (Lot Number 0902951), and Rhinovirus type 14 (ATCC# VR-284).

TABLE 68 Reduction of Infectivity Test Product: Methylsulfonylmethane,6% (lot# 0902951) Virus: Rhinovirus type 14 strain 1059 ATCC # VR-284Host Cell Line: MRC-5 Host Cell Line ATCC # CCL-171 Dilutions VirusControl Test Product Cell (−log₁₀) Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2Rep. 3 Control 0000 −2 NT NT NT ++++ ++++ ++++ −3 ++++ ++++ ++++ ++++++++ ++++ −4 ++++ ++++ ++++ ++++ ++++ ++++ −5 ++++ ++++ ++++ +000 +0+0+0++ −6 0000 0000 0+0+ 0000 0000 0000 −7 0000 0000 0000 0000 0000 0000TCID₅₀ 5.50log₁₀ 5.50log₁₀ 6.00log₁₀ 4.75log₁₀ 5.00log₁₀ 5.25log₁₀Average 5.67log₁₀ 5.00log₁₀ TCID₅₀ Log 0.92log₁₀ 0.67log₁₀ 0.42log₁₀Reduction Average 0.67log₁₀ Log Reduction Percent 87.98% 78.62% 61.98%Reduction Average 78.62% Percent Reduction + = CPE Present 0 = CPE notdetected NT = Not Tested Rep = Replicate * - Log Reduction = AverageTCID₅₀ of Virus Control − TCID₅₀ of the Test Replicate ** - Average %Reduction (calculated from average log₁₀ reduction) = 100 − (1/TCID₅₀Reduction) * 100

TABLE 69 Reduction of Infectivity Test Product: Methylsulfonylmethane,5% (lot# 0902951) Virus: Rhinovirus type 14 strain 1059 ATCC # VR-284Host Cell Line: MRC-5 Host Cell Line ATCC # CCL-171 Dilutions VirusControl Test Product Cell (−log₁₀) Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2Rep. 3 Control 0000 −2 NT NT NT ++++ ++++ ++++ −3 ++++ ++++ ++++ ++++++++ ++++ −4 ++++ ++++ ++++ ++++ ++++ ++++ −5 ++++ ++++ ++++ 0+++ ++++++++ −6 0000 0000 0+0+ 0000 0000 +00+ −7 0000 0000 0000 0000 0000 0000TCID₅₀ 5.50log₁₀ 5.50log₁₀ 6.00log₁₀ 5.25log₁₀ 5.50log₁₀ 6.00log₁₀Average 5.67log₁₀ 5.58log₁₀ TCID₅₀ Log 0.09log₁₀ 0.17log₁₀ 0.00log₁₀Reduction Average 0.09log₁₀ Log Reduction Percent 18.72% 32.39% 00.00%Reduction Average 18.72% Percent Reduction + = CPE Present 0 = CPE notdetected NT = Not Tested Rep = Replicate * - Log Reduction = AverageTCID₅₀ of Virus Control − TCID₅₀ of the Test Replicate ** - Average %Reduction (calculated from average log₁₀ reduction) = 100 − (1/TCID₅₀Reduction) * 100

TABLE 70 Reduction of Infectivity Test Product: Methylsulfonylmethane,4% (lot# 0902951) Virus: Rhinovirus type 14 strain 1059 ATCC # VR-284Host Cell Line: MRC-5 Host Cell Line ATCC # CCL-171 Dilutions VirusControl Test Product Cell (−log₁₀) Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2Rep. 3 Control 0000 −2 NT NT NT ++++ ++++ ++++ −3 ++++ ++++ ++++ ++++++++ ++++ −4 ++++ ++++ ++++ ++++ ++++ ++++ −5 ++++ ++++ ++++ 00++ ++++++++ −6 0000 0000 0+0+ 0000 0000 0+00 −7 0000 0000 0000 0000 0000 0000TCID₅₀ 5.50log₁₀ 5.50log₁₀ 6.00log₁₀ 5.00log₁₀ 5.50log₁₀ 5.75log₁₀Average 5.67log₁₀ 5.42log₁₀ TCID₅₀ Log 0.67log₁₀ 0.17log₁₀ 0.00log₁₀Reduction Average 0.28log₁₀ Log Reduction Percent 78.62% 32.39% 00.00%Reduction Average 47.52% Percent Reduction + = CPE Present 0 = CPE notdetected NT = Not Tested Rep = Replicate * - Log Reduction = AverageTCID₅₀ of Virus Control − TCID₅₀ of the Test Replicate ** - Average %Reduction (calculated from average log₁₀ reduction) = 100 − (1/TCID₅₀Reduction) * 100

TABLE 71 Reduction of Infectivity Test Product: Methylsulfonylmethane,3% (lot# 0902951) Virus: Rhinovirus type 14 strain 1059 ATCC # VR-284Host Cell Line: MRC-5 Host Cell Line ATCC # CCL-171 Dilutions VirusControl Test Product Cell (−log₁₀) Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2Rep. 3 Control 0000 −2 NT NT NT ++++ ++++ ++++ −3 ++++ ++++ ++++ ++++++++ ++++ −4 ++++ ++++ ++++ ++++ ++++ ++++ −5 ++++ ++++ ++++ ++0+ ++++++++ −6 0000 0000 0+0+ 000+ 0000 000+ −7 0000 0000 0000 0000 0000 +000TCID₅₀ 5.50log₁₀ 5.50log₁₀ 6.00log₁₀ 5.50log₁₀ 5.50log₁₀ 6.00log₁₀Average 5.67log₁₀ 5.67log₁₀ TCID₅₀ Log 0.17log₁₀ 0.17log₁₀ 0.00log₁₀Reduction Average 0.11log₁₀ Log Reduction Percent 32.39% 32.39% 00.00%Reduction Average 22.38% Percent Reduction + = CPE Present 0 = CPE notdetected NT = Not Tested Rep = Replicate * - Log Reduction = AverageTCID₅₀ of Virus Control − TCID₅₀ of the Test Replicate ** - Average %Reduction (calculated from average log₁₀ reduction) = 100 − (1/TCID₅₀Reduction) * 100

TABLE 72 Reduction of Infectivity Test Product: Methylsulfonylmethane,2% (lot# 0902951) Virus: Rhinovirus type 14 strain 1059 ATCC # VR-284Host Cell Line: MRC-5 Host Cell Line ATCC # CCL-171 Dilutions VirusControl Test Product Cell (−log₁₀) Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2Rep. 3 Control 0000 −2 NT NT NT ++++ ++++ ++++ −3 ++++ ++++ ++++ ++++++++ ++++ −4 ++++ ++++ ++++ ++++ ++++ ++++ −5 ++++ ++++ ++++ ++++ 0+++0000 −6 0000 0000 0+0+ 0000 00+0 00+0 −7 0000 0000 0000 0000 0000 0000TCID₅₀ 5.50log₁₀ 5.50log₁₀ 6.00log₁₀ 5.50log₁₀ 5.50log₁₀ 4.75log₁₀Average 5.67log₁₀ 5.25log₁₀ TCID₅₀ Log 0.17log₁₀ 0.17log₁₀ 0.92log₁₀Reduction Average 0.42log₁₀ Log Reduction Percent 32.39% 32.39% 87.98%Reduction Average 61.98% Percent Reduction + = CPE Present 0 = CPE notdetected NT = Not Tested Rep = Replicate * - Log Reduction = AverageTCID₅₀ of Virus Control − TCID₅₀ of the Test Replicate ** - Average %Reduction (calculated from average log₁₀ reduction) = 100 − (1/TCID₅₀Reduction) * 100

TABLE 73 Reduction of Infectivity Test Product: Methylsulfonylmethane,1% (lot# 0902951) Virus: Rhinovirus type 14 strain 1059 ATCC # VR-284Host Cell Line: MRC-5 Host Cell Line ATCC # CCL-171 Dilutions VirusControl Test Product Cell (−log₁₀) Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2Rep. 3 Control 0000 −2 NT NT NT ++++ ++++ ++++ −3 ++++ ++++ ++++ ++++++++ ++++ −4 ++++ ++++ ++++ ++++ ++++ ++++ −5 ++++ ++++ ++++ ++++ ++0+++++ −6 0000 0000 0+0+ +0+0 0000 000+ −7 0000 0000 0000 0000 0000 0000TCID₅₀ 5.50log₁₀ 5.50log₁₀ 6.00log₁₀ 6.00log₁₀ 5.25log₁₀ 5.75log₁₀Average 5.67log₁₀ 5.67log₁₀ TCID₅₀ Log 0.00log₁₀ 0.42log₁₀ 0.00log₁₀Reduction Average 0.14log₁₀ Log Reduction Percent 00.00% 61.98% 00.00%Reduction Average 27.56% Percent Reduction + = CPE Present 0 = CPE notdetected NT = Not Tested Rep = Replicate * - Log Reduction = AverageTCID₅₀ of Virus Control − TCID₅₀ of the Test Replicate ** - Average %Reduction (calculated from average log₁₀ reduction) = 100 − (1/TCID₅₀Reduction) * 100

TABLE 74 Reduction of Infectivity Test Product: Methylsulfonylmethane,0.5% (lot# 0902951) Virus: Rhinovirus type 14 strain 1059 ATCC # VR-284Host Cell Line: MRC-5 Host Cell Line ATCC # CCL-171 Dilutions VirusControl Test Product Cell (−log₁₀) Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2Rep. 3 Control 0000 −2 NT NT NT ++++ ++++ ++++ −3 ++++ ++++ ++++ ++++++++ ++++ −4 ++++ ++++ ++++ ++++ ++++ ++++ −5 ++++ ++++ ++++ ++++ ++++0000 −6 0000 0000 0+0+ 0000 +000 0000 −7 0000 0000 0000 0000 0000 0000TCID₅₀ 5.50log₁₀ 5.50log₁₀ 6.00log₁₀ 5.50log₁₀ 5.75log₁₀ 4.50log₁₀Average 5.67log₁₀ 5.25log₁₀ TCID₅₀ Log 0.17log₁₀ 0.00log₁₀ 1.17log₁₀Reduction Average 0.40log₁₀ Log Reduction Percent 32.39% 00.00% 93.24%Reduction Average 60.19% Percent Reduction + = CPE Present 0 = CPE notdetected NT = Not Tested Rep = Replicate * - Log Reduction = AverageTCID₅₀ of Virus Control − TCID₅₀ of the Test Replicate ** - Average %Reduction (calculated from average log₁₀ reduction) = 100 − (1/TCID₅₀Reduction) * 100

Table 75 presents the Virus Control infectivity (TCID₅₀), the averageinfectivity (TCID₅₀), and the log₁₀ and percent enhancement observed inPre-treatment test of the Test Product, Methylsulfonylmethane (LotNumber 0902951), and Swine-like H1N1 Influenza A virus strainA/California/04/2009 (CDC ID #2009712047).

TABLE 75 Enhancement of Infectivity Test Product: Methylsulfonylmethane,3% (lot# 0902951) Virus: Swine-like Influenza A H1N1 strainA/California/04/2009 CDC ID # 2009712047 Host Cell Line: MDCK Host CellLine ATCC # CCL-34 Dilutions Virus Control Test Product (−log₁₀) Rep. 1Rep. 2 Rep. 3 Rep. 1 Rep. 2 Rep. 3 Cell Control 0000 −2 NT NT NT ++++++++ ++++ −3 ++++ ++++ ++++ ++++ ++++ ++++ −4 ++++ ++++ ++++ ++++ ++++++++ −5 00+0 +000 +0+0 00++ +00+ 0++0 −6 0000 0000 0000 0000 0000 0000−7 0000 0000 0000 0000 0000 0000 TCID₅₀ 4.75log₁₀ 4.75log₁₀ 5.00log₁₀5.00log₁₀ 5.00log₁₀ 5.00log₁₀ Average 4.83log₁₀ 5.00log₁₀ TCID₅₀ Log0.17log₁₀ 0.17log₁₀ 0.17log₁₀ Stimulation Average 0.17log₁₀ LogStimulation Percent 32.39% 32.39% 32.39% Stimulation Average 32.39%Percent Stimulation + = CPE Present 0 = CPE not detected NT = Not TestedRep = Replicate * - Log Stimulation = Average TCID₅₀ of Test − TCID₅₀ ofReplicate of the Virus Control ** - Average % Stimulation (calculatedfrom average log₁₀ stimulation) = 100 − (1/TCID₅₀ Stimulation) * 100

Tables 76 through 78 the Virus Control infectivity (TCID₅₀), the averageinfectivity (TCID₅₀), and the log₁₀ and percent enhancement observed inPre-treatment test of the Test Product, Methylsulfonylmethane (LotNumber 0902951), and Rhinovirus type 14 (ATCC # VR-284).

TABLE 76 Enhancement of Infectivity Test Product: Methylsulfonylmethane,5% (lot# 0902951) Virus: Rhinovirus type 14 strain 1059 ATCC # VR-284Host Cell Line: MRC-5 HostCell Line ATCC # CCL-171 Dilutions VirusControl Test Product Cell (−log₁₀) Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2Rep. 3 Control 0000 −2 NT NT NT ++++ ++++ ++++ −3 ++++ ++++ ++++ ++++++++ ++++ −4 ++++ ++++ ++++ ++++ ++++ ++++ −5 ++++ ++++ ++++ 0+++ ++++++++ −6 0000 0000 0+0+ 0000 0000 +00+ −7 0000 0000 0000 0000 0000 0000TCID₅₀ 5.50log₁₀ 5.50log₁₀ 6.00log₁₀ 5.25log₁₀ 5.50log₁₀ 6.00log₁₀Average 5.67log₁₀ 5.58log₁₀ TCID₅₀ Log 0.08log₁₀ 0.08log₁₀ 0.00log₁₀Stimulation Average Log 0.053log₁₀ Stimulation Percent 16.82% 16.82%00.00% Stimulation Average 11.49% Percent Stimulation + = CPE Present 0= CPE not detected NT = Not Tested Rep = Replicate * - Log Stimulation =Average TCID₅₀ of Test − TCID₅₀ of Replicate of the Virus Control ** -Average % Stimulation (calculated from average log₁₀ stimulation) = 100− (1/TCID₅₀ Stimulation) * 100

TABLE 77 Enhancement of Infectivity Test Product: Methylsulfonylmethane,3% (lot# 0902951) Virus: Rhinovirus type 14 strain 1059 ATCC # VR-284Host Cell Line: MRC-5 Host Cell Line ATCC # CCL-171 Dilutions VirusControl Test Product Cell (−log₁₀) Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2Rep. 3 Control 0000 −2 NT NT NT ++++ ++++ ++++ −3 ++++ ++++ ++++ ++++++++ ++++ −4 ++++ ++++ ++++ ++++ ++++ ++++ −5 ++++ ++++ ++++ ++0+ ++++++++ −6 0000 0000 0+0+ 000+ 0000 000+ −7 0000 0000 0000 0000 0000 +000TCID₅₀ 5.50log₁₀ 5.50log₁₀ 6.00log₁₀ 5.50log10 5.50log10 6.00log10Average 5.67log₁₀ 5.67log₁₀ TCID₅₀ Log 0.17log₁₀ 0.17log₁₀ 0.00log₁₀Stimulation Average 0.11log₁₀ Log Stimulation Percent 32.39% 32.39%00.00% Stimulation Average 22.38% Percent Stimulation + = CPE Present NT= Not Tested 0 = CPE not detected Rep = Replicate * - Log Stimulation =Average TCID₅₀ of Test − TCID₅₀ of Replicate of the Virus Control ** -Average % Stimulation (calculated from average log₁₀ stimulation) = 100− (1/TCID₅₀ Stimulation) * 100

TABLE 78 Enhancement of Infectivity Test Product: Methylsulfonylmethane,1% (lot# 0902951) Virus: Rhinovirus type 14 strain 1059 ATCC # VR-284Host Cell Line: MRC-5 HostCell Line ATCC # CCL-171 Dilutions VirusControl Test Product Cell (−log₁₀) Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2Rep. 3 Control 0000 −2 NT NT NT ++++ ++++ ++++ −3 ++++ ++++ ++++ ++++++++ ++++ −4 ++++ ++++ ++++ ++++ ++++ ++++ −5 ++++ ++++ ++++ ++++ ++0+++++ −6 0000 0000 0+0+ +0+0 0000 000+ −7 0000 0000 0000 0000 0000 0000TCID₅₀ 5.50log₁₀ 5.50log₁₀ 6.00log₁₀ 6.00log₁₀ 5.25log₁₀ 5.75log₁₀Average 5.67log₁₀ 5.67log₁₀ TCID₅₀ Log 0.17log₁₀ 0.17log₁₀ 0.00log₁₀Stimulation Average Log 0.11log₁₀ Stimulation Percent 32.39% 32.39%00.00% Stimulation Average 22.38% Percent Stimulation + = CPE Present 0= CPE not detected NT = Not Tested Rep = Replicate * - Log Stimulation =Average TCID₅₀ of Test − TCID₅₀ of Replicate of the Virus Control ** -Average % Stimulation (calculated from average log₁₀ stimulation) = 100− (1/TCID₅₀ Stimulation) * 100

Nonlinear Regression, Dose Versus Response:

Dose-Response (Inhibition) Analyses were performed for the test productconcentrations converted into mM (test product molecular weight=94.13).Nonlinear regression analyses were as follows: log (inhibitor) vs.normalized response−Variable slope. Concentrations are presented inTable 79.

TABLE 79 Concentration, % Concentration, mM 7% 74.365 6% 63.742 5%53.118 4% 42.494 3% 31.871 2% 21.247 1% 10.624 0.5%  5.312Table 80 presents the data input for Herpes Simplex Virus.

TABLE 80 Dose, mM Response, % reduction 74.365 99.320 99.790 99.79063.742 87.980 87.980 93.240 53.118 87.980 93.240 98.800 42.494 98.80093.240 97.860 31.871 96.200 93.240 61.980 21.247 61.980 93.240 87.98010.624 78.620 78.620 78.620 5.312 32.390 61.980 32.390

Table 81 presents transform (log of dose=X=Log(X)) of data for HerpesSimplex Virus.

TABLE 81 Dose, mM Response, % reduction 1.871369 99.320 99.790 99.7901.804426 87.980 87.980 93.240 1.725242 87.980 93.240 98.800 1.62832898.800 93.240 97.860 1.503396 96.200 93.240 61.980 1.327298 61.98093.240 87.980 1.026288 78.620 78.620 78.620 0.7252581 32.390 61.98032.390

Table 82 presents of transform of normalize of the data for HerpesSimplex Virus. The percent reduction was normalized as follows: 32.39%becomes 0% for all data set; 99.79% becomes 100% for all data set.

TABLE 82 Dose, mM Response, % reduction 1.871369 99.30267 100.000100.000 1.804426 82.47775 82.47775 90.2819 1.725242 82.47775 90.281998.53116 1.628328 98.53116 90.2819 97.1365 1.503396 94.67358 90.281943.90208 1.327298 43.90208 90.2819 82.47775 1.026288 68.59051 68.5905168.59051 0.7252581 0.000 43.90208 0.000

IC₅₀ computation for Herpes Simplex Virus is presented in Table 83. Thebest fit value for Herpes Simplex Virus IC₅₀ was determined 10.13 mM.However, due to a significant variation in virus reduction, IC₅₀ valuesranging from 7.144 mM to 14.37 mM can be considered a more plausibleapproximation.

TABLE 83 log(inhibitor) vs. normalized response -- Variable slopeBest-fit values LogIC50 1.006 HillSlope 1.523 IC50 10.13 Std. ErrorLogIC50 0.07314 HillSlope 0.3281 95% Confidence Intervals LogIC50 0.8539to 1.157 HillSlope 0.8428 to 2.204 IC50  7.144 to 14.37 Goodness of FitDegrees of Freedom 22 R square 0.6761 Absolute Sum of Squares 6312 Sy.x16.94 Number of points Analyzed 24Table 84 presents the data input for Swine-like Influenza virus A H1N1.

TABLE 84 Dose, mM Response, % reduction 74.365 91.680 91.680 85.21063.742 91.680 91.680 85.210 53.118 53.230 53.230 73.700 42.494 16.82053.230 16.820 31.871 0.000 0.000 0.000 21.247 53.230 16.820 85.21010.624 53.230 16.820 53.230 5.312 91.680 53.230 73.700

Table 85 presents transform [log of dose=X=Log(X)] of data forSwine-like Influenza virus A H1N1.

TABLE 85 Dose, mM Response, % reduction 1.871369 91.680 91.680 85.2101.804426 91.680 91.680 85.210 1.725242 53.230 53.230 73.700 1.62832816.820 53.230 16.820 1.503396 0.000 0.000 0.000 1.327298 53.230 16.82085.210 1.026288 53.230 16.820 53.230 0.7252581 91.680 53.230 73.700

Table 86 presents of transform of normalize of the data for Swine-likeInfluenza virus A H1N1. The percent reduction was normalized as follows:0% becomes 0% for all data set; 91.68% becomes 100% for all data set.

TABLE 86 Dose, mM Response, % reduction 1.871369 100.000 100.00092.94284 1.804426 100.000 100.000 92.94284 1.725242 58.06065 58.0606580.38831 1.628328 18.34642 58.06065 18.34642 1.503396 0.000 0.000 0.0001.327298 58.06065 18.34642 92.94284 1.026288 58.06065 18.34642 58.060650.7252581 100.000 58.06065 80.38831

IC₅₀ computation for Swine-like Influenza virus A H1N1 is presented inTable 87. The best-fit IC₅₀ value for Swine-like Influenza virus A H1N1was determined 5.114 mM. IC₅₀ values with 95% confidence intervalsranged from 0.008038 mM to 3253 mM. In view of inconsequence of virusreduction (U-shaped curve) MSM IC₅₀s were determined with a significantapproximation. IC₉₀ values can not be concluded from this data set.

TABLE 87 log(inhibitor) vs. normalized response -- Variable slopeBest-fit values LogIC50 0.7087 HillSlope 0.2135 IC50 5.114 Std. ErrorLogIC50 1.352 HillSlope 0.3534 95% Confidence Intervals LogIC50 −2.095to 3.512 HillSlope −0.5194 to 0.9464 IC50 0.008038 to 3253   Goodness ofFit Degrees of Freedom 22 R square 0.01810 Absolute Sum of Squares 29296Sy.x 36.49 Number of points Analyzed 24Table 88 presents the data input for Rhinovirus type 14.

TABLE 88 Dose, mM Response, % reduction 74.365 96.200 93.240 93.24063.742 87.980 78.620 61.980 53.118 18.720 32.390 0.000 42.494 78.62032.390 0.000 31.871 32.390 32.390 0.000 21.247 32.390 32.390 87.98010.624 0.000 61.980 0.000 5.312 32.390 0.000 93.240

Table 89 presents transform [log of dose=X=Log(X)] of data forRhinovirus type 14.

TABLE 89 Dose, mM Response, % reduction 1.871369 96.200 93.240 93.2401.804426 87.980 78.620 61.980 1.725242 18.720 32.390 0.000 1.62832878.620 32.390 0.000 1.503396 32.390 32.390 0.000 1.327298 32.390 32.39087.980 1.026288 0.000 61.980 0.000 0.7252581 32.390 0.000 93.240

Table 90 presents of transform of normalize of the data for Rhinovirustype 14. The percent reduction was normalized as follows: 0% becomes 0%for all data set; 96.20% becomes 100% for all data set.

TABLE 90 Dose, mM Response, % reduction 1.871369 100.000 96.9230896.92308 1.804426 91.45531 81.72558 64.42828 1.725242 19.45946 33.669440.000 1.628328 81.72558 33.66944 0.000 1.503396 33.66944 33.66944 0.0001.327298 33.66944 33.66944 91.45531 1.026288 0.000 64.42828 0.0000.7252581 33.66944 0.000 96.92308

IC₅₀ computation for Rhinovirus type 14 is presented in Table 91. Thebest fit IC₅₀ value for Rhinovirus type 14 was determined 38.16 mM. IC₅₀values with 95% confidence intervals ranged from 13.07 mM to 111.4 mM.In view of inconsequence of virus reduction (U-shaped curve) MSM IC₅₀swere determined with a significant approximation. IC₉₀ values cannot beconcluded from this data set.

TABLE 91 log(inhibitor) vs. normalized response -- Variable slopeBest-fit values LogIC50 1.582 HillSlope 0.6280 IC50 38.16 Std. ErrorLogIC50 0.2244 HillSlope 0.4179 95% Confidence Intervals LogIC50 1.116to 2.047 HillSlope −0.2387 to 1.495  IC50 13.07 to 111.4 Goodness of FitDegrees of Freedom 22 R square 0.1044 Absolute Sum of Squares 29118 Sy.x36.38 Number of points Analyzed 24

Example 24 Effect of MSM on Algae

This example shows effects of MSM on algae activity.

Two species of Chlorella were examined for growth—Chlorella sorokinianaa freshwater species and Chlorella minutissima a marine species. Thestudy measured the effect of algal growth in a freshwater and saltwaterenvironment with the addition of MSM in which MSM was added at thefollowing concentrations: 0%, 0.25%, 2%, 5%, 10% and 20%. Growth wasmeasured on day 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. The growth curvesof percent transmittance of the algae were compared between the MSMconcentrations with the 0% MSM concentration as a sample control foreach microorganism. The MSM stock powder was supplied by BergstromNutrition with certificate of analysis. The powder was the microprillformula, lot #0806809. All media, water and stock MSM powder wassterility checked prior to the study. The following media were purchasedfrom UTEX Culture Collection of Algae: Enriched Salt Water Medium andVolvox Dextrose Medium.

The algae were grown for 48 hours in the appropriate medium. The initialsuspension was enumerated for each alga and is referred to as thestarting inoculums. Chlorella sorokiniana was at 381 million cells permilliliter and Chlorella minutissima was at 19 million cells permilliliter. One milliliter of the algal solution was placed into 9 mLsof medium and mixed by vortexing. This was repeated for eachconcentration of MSM medium mixture. The tube of algae and MSM wereincubated at room temperature with exposure to sunlight. The working MSMconcentrations were prepared from a single 20.0% MSM solution and werediluted accordingly with medium to get the desired final concentrationof MSM. All solutions were verified for sterility before proceeding withthe study. Each dilution of MSM for each organism was set up in andanalyzed in triplicate for each time interval measured. The samples weremeasured by percent transmittance on a UV/VIS spectrophotometer atwavelength 750 nm. The medium stock was tested for percent transmittancebackground levels at every time interval were measured. The results fromthese studies are provided in the Tables 92 and 93 below. The lowerpercent transmittance indicated a higher growth factor. These studiesdemonstrate that MSM treatment can increase growth of algae.

TABLE 92 Growth of Chlorella sorokiniana MSM Concentrations 0 0.5 1 2.55 10 20 Medium Sample 0 80.3 47.3 48.7 40.6 35.6 31.2 19.3 30.2 Day 150.6 41.8 42.5 43.1 31.2 31.3 21.9 40.3 2 24.5 29.3 37.5 44.9 27.4 29.724.1 82.4 3 29.9 29.6 37.5 43.8 21.4 28.4 28.1 91.2 4 25.4 19.0 17.326.1 18.2 29.0 31.4 94.5 5 10.6 12.4 13.5 10.3 15.7 28.7 33.6 93.3 610.5 12.5 13.0 10.9 15.9 27.1 36.8 97.8 7 10.0 11.9 12.5 11.0 16.3 27.640.6 34.3 8 10.0 12.2 12.4 11.2 17.0 27.1 40.8 32.2 9 8.5 7.5 7.8 8.413.7 76.4 84.8 30.4 10 7.4 6.4 6.6 6.0 12.8 93.8 96.7 18.8

TABLE 93 Growth of Chlorella minutissima MSM Percentage 0.0 0.5 1.0 2.55.0 10.0 20.0 Medium Sample 0 72.4 93.5 91.1 82.8 68.9 49.6 30.0 105.1Day 1 74.6 70.1 80.5 75.1 87.4 49.2 33.8 105.1 2 51.5 45.4 40.0 59.576.5 50.8 51.1 105.1 3 33.4 32.1 31.0 45.4 62.0 51.9 54.8 105.1 4 28.227.6 27.9 33.6 52.9 52.0 57.3 105.1 5 26.4 26.6 26.5 32.4 52.2 51.9 57.5105.1 6 25.6 25.1 25.4 30.0 50.7 54.9 57.4 105.1 7 24.3 23.6 24.4 28.651.0 56.1 57.1 106.4 8 24.1 22.8 23.7 27.7 51.9 58.6 55.8 107.0 9 18.020.4 21.0 23.3 47.1 41.4 45.1 109.3 10 14.9 18.9 19.4 21.1 44.1 36.730.2 112.0

Example 25 Absorption of MSM in Topical Formulation is within RecognizedSafe Levels

This example shows absorption of MSM in topical formulations is withinrecognized safe levels.

New Zealand White rabbits, which are an accepted animal model for dermalabsorption studies, were used to assess the absorption and resultantblood levels of MSM. Rabbits were obtained from Charles River Canada(Saint-Constant, Quebec). Five male rabbits, ages 12-13 weeks andranging in weight from 2.6 kg to 2.7 kg were used for the dermalabsorption studies. Rabbits were used because of their greater skinpermeability as compared to rats, pigs or humans. Thus, testing onrabbits is a more conservative approach for the safety of topicalproducts for human use. The size of rabbit was based on the ethicalrestriction of collecting greater than 6 mL/kg body weight of bloodwithin a two week period. The total volume of blood to be removed duringthis study was 10 mL on a single day. One animal per group was used tominimize the number of animals required Animals were housed individuallyin stainless steel cages with 12 hours light/dark cycles. The animalroom environment was monitored daily (targeted ranges: 18-26° C. andrelative humidity 25-50%). Fresh air was supplied to the room at asufficient rate to provide approximately 15 to 17 changes of room airper hour. Clinical observations were conducted for all animals to ensureanimals were in good health prior to dosing. Morbidity and mortalityobservations were also conducted during the study period.

Treatment groups were as shown in Table 94.

TABLE 94 Study 1 Design Surface Area Volume Number of Blood CollectionGroup Test Article Exposed Applied Animals Times (min) A 10% MSM + 90%Water 6 cm² 0.5 mL 1 0 (pre-dose), 10, 30, 120, 480 minutes B 50% DMSO +50% Water 6 cm² 0.5 mL 1 0 (pre-dose), 10, 30, 120, 480 minutes C 70%DMSO + 30% Water 6 cm² 0.5 mL 1 0 (pre-dose), 10, 30, 120, 480 minute D10% MSM + 50% DMSO + 6 cm² 0.5 mL 1 0 (pre-dose), 10, 30, 40% Water 120,480 minutes E 10% MSM + 70% DMSO + 6 cm² 0.5 mL 1 0 (pre-dose), 10, 30,20% Water 120, 480 minutes

One day prior to the study, the rump of each rabbit was closely clippedusing hair clippers. An area of 6 cm² was measured and marked to ensureequivalence in the application of the various compositions. Each productwas applied by pipetting 0.5 mL of each composition into the center ofthe test area and spread to cover the entire test area. After the 5minute exposure period, the compositions were removed by wiping, rinsingand drying the test area.

Prior to blood collection, animals were tranquilized with Acepromazine(1 mg/kg) by intramuscular injection in the right hind leg muscle, afterwhich EMLA cream (lidocaine/prilocaine) was applied to both ears alongthe ear artery. Blood was collected by insertion of a 21 G needle (hubremoved) into the ear artery. Approximately 2 mL of whole blood wascollected into 4 mL vacuutainer tubes (Becton Dickinson, Mississauga,ON) containing K₂EDTA. Tubes were inverted to mix with the anticoagulantand stored refrigerated until plasma was separated by centrifugation.Plasma was separated from whole blood by centrifugation at 3000×g for 10minutes. Plasma was collected, transferred and stored in a cryovial at−70° C. until further processing for MSM analysis.

Following the 5 minute exposure period to the various test products (seeTable 1), blood was collected after 10 minutes, 30 minutes, 2 hours and8 hours. Prior to the 2 and 8 hour blood collections, EMLA cream wasapplied to the ears (approximately 30 minutes prior to each of theseblood draws) as the anesthetic effects of the EMLA cream lastsapproximately 1 to 2 hours. Both EMLA cream and Acepromazine were useddue to ethical considerations and to provide for the well being of theanimals used in this study.

The concentrations of MSM in plasma were quantified by gaschromatography-mass spectrometry (GC/MS) based on established methods.Briefly, 450 μL of plasma sample was mixed with 50 μL of physiologicalsaline and vortexed for 30 seconds. Following this 1 mL of Acetonitrile(Fisher, HPLC grade) was added to the mixture. The solution was vortexedvigorously for 60 seconds and centrifuged at 2000 rpm for 5 minutes. Onemicroliter of the clear supernatant was introduced to the GC/MS system(GC/MS QP20108 EI, Shimadzu, Kyoto, Japan). The analysis was performedon a 8himadzu SHR5XLB column (0.25 mm ID X length 30 m, film 0.25 um,Kyoto, Japan). The retention time of MSM was 6.1-6.3 minutes. MSM wasdetected with MS and m/z 79 (M+−15) was used for monitoring MSM ion SIMprofiles. Helium gas was used as the carrier gas, head pressure was 0.25kg/cm2, make-up gas was 30 mL/min, column temperature was 80° C.,injector temperature 120° C., separator temperature 200° C. and ionsource temperature 250° C. The ionization energy was 70 eV. An externalstandard graph was prepared with MSM dissolved in acetonitrile at thefollowing concentrations: 62.5 μg/ml, 31.3 μg/ml, 15.6 μg/ml, 7.8 μg/ml,3.9 μg/ml, 1.9 μg/ml, 0.98 μg/ml and 0.49 μg/ml. The MSM concentrationin plasma samples was calculated from the slope of the standard curve.The best fitted graph was linear with a R2 value of 0.998.

All animals were observed prior to the start of the study and alldemonstrated good health. During the course of the study and subsequentto the study, all animals demonstrated good health. Morbidity, mortalityand injury were assessed twice daily. No animals demonstrated anymorbidity, mortality or injury.

The results of the absorption study are summarized in Table 95. Baselineplasma concentrations of MSM (prior to exposure to test articles) rangedbetween 4.2 μg/mL and 104.2 μg/mL. The variation in baseline is withinthe normal range of variation of natural MSM concentrations that havebeen established in prior studies. Following exposure to the varioustest articles, the highest plasma concentrations of MSM measured wereless than or equal to approximately 140 μg/mL. This peak concentrationresults from exposure to 10% MSM+70% DMSO+20% water. When corrected fornatural variation in baseline MSM concentrations, the largest change inplasma MSM was detected in the 70% DMSO+30% water group. These datasuggest that variations in MSM, either due to absorption or due tometabolism of DMSO, are within the natural range of MSM concentrations.

TABLE 95 Concentration of MSM in Plasma After Exposure to MSM and DMSOTime point MSM Concentration Treatment (minute) (μg/mL) 10% MSM + 0 25.690% water 10 17.6 30 16.3 120 14.0 480 15.4 50% DMSO + 0 4.2 50% water10 6.9 30 6.9 120 7.4 480 12.6 70% DMSO + 0 56.7 30% water 10 89.0 3098.9 120 128.7 480 120.2 10% MSM + 0 104.2 50% DMSO + 10 116.5 40% water30 127.9 120 128.4 480 140.4 10% MSM + 0 26.8 70% DMSO + 10 37.3 20%water 30 30.9 120 33.9 480 44.4

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

1. A method of enhancing fermentation efficiency of a microorganism, themethod comprising: contacting medium containing a microorganism capableof fermentation with methylsulfonylmethane (MSM), wherein the MSM isprovided at a concentration of about 0.5% to about 5% by weight of themedium or at a concentration of about 0.5% to about 5% by weight of themoisture content of the medium, wherein the MSM increases thefermentation efficiency of the microorganism as compared to thefermentation efficiency in the absence of MSM.
 2. The method of claim 1,wherein enhancing fermentation efficiency comprises an at least 50%increase in alcohol, carbon dioxide or acid production in the presenceof MSM by the microorganism as compared to alcohol or acid production inthe absence of MSM.
 3. The method of claim 1, wherein the method ofenhancing fermentation efficiency is for the production of beer, cider,wine, a biofuel, bread, dairy product or any combination thereof.
 4. Themethod of claim 2, wherein enhancing fermentation efficiency comprisesan at least 50% increase in production of ethanol, methanol or acombination of thereof as compared to production of ethanol, methanol ora combination of thereof in the absence of MSM.
 5. The method of claim1, wherein the microorganism is yeast and the method of enhancingfermentation is for the production of beer.
 6. The method of claim 1,wherein the microorganism is algae and the method of enhancingfermentation is for the production of biofuel.
 7. The method of claim 2,wherein enhancing fermentation efficiency comprises an at least 50%increase in carbon dioxide production in the presence of MSM by themicroorganism as compared to carbon dioxide production in the absence ofMSM, the microorganism is yeast and the method of enhancing fermentationis for the production of bread.
 8. The method of claim 2, whereinenhancing fermentation efficiency comprises an at least 50% increase inlactic acid production in the presence of MSM by the microorganism ascompared to lactic acid production in the absence of MSM and the methodof enhancing fermentation is for the production of a dairy product. 9.The method of claim 1, wherein the concentration of MSM is about 0.5%.10. The method of claim 1, wherein the medium comprises a sodiumchloride concentration of less than 5% of total moisture content.
 11. Anin vitro method for enhancing the growth of one or more probioticmicroorganisms, the method comprising: contacting one or more probioticmicroorganisms with a medium capable of supporting growth of one or moreprobiotic microorganisms; and providing methylsulfonylmethane (MSM) tothe medium at about 0.4% to about 5% by weight of the medium or byweight of a moisture content of the medium thereby enhancing growth ofthe one or more microorganisms in vitro as compared to growth of the oneor more microorganisms in vitro in the absence of MSM.
 12. The method ofclaim 11, wherein the concentration of MSM is about 1% to about 3% ofthe weight of the medium or the moisture content of the medium.
 13. Themethod of claim 11, wherein the one or more probiotic microorganismscomprises Lactobacillus acidophilus, Lactobacillus delbrueckii, Bacilluscoagulans, Lactobacillus rhamnosus, Bifidobacteruim bifidum or anycombination thereof.
 14. The method of claim 11, wherein the mediumcomprises a probiotic-containing product, such as milk, yogurt, riceyogurt, frozen yogurt, chocolate, cheese, beer, wine, vinegar,sauerkraut or any combination thereof.
 15. A method for enhancing growthof a microorganism in a diagnostic test sample, the method comprising:contacting the diagnostic test sample comprising one or moremicroorganisms with a medium capable of supporting growth of the one ormore microorganisms; providing (methylsulfonylmethane) MSM to the mediumat a concentration of about 0.4% to about 5% by weight of the medium orby weight of a moisture content of the medium, thereby enhancing thegrowth of the one or more microorganisms in the diagnostic test sampleas compared to growth of the one or more microorganisms in the absenceof MSM.
 16. A method of inhibiting microbial activity, the methodcomprising: selecting a medium that is susceptible to H1N1 influenzacontamination; and contacting the medium with methylsulfonylmethane(MSM) at a concentration of about 10% to about 16% of weight by volume,thereby inhibiting H1N1 influenza microbial activity.
 17. The method ofclaim 16, wherein the medium comprises a bodily fluid, a bodily tissue,or a surface.
 18. The method of claim 16, wherein contacting the mediumcomprises spraying or wiping the medium susceptible to microbialcontamination with MSM.
 19. The method of claim 18, wherein the surfaceis a household surface, bedding, coverings, industrial equipment orsurface, blood, skin or a combination thereof.
 20. The method of claim16, wherein MSM is provided in a composition, wherein said compositionis free of bleach or free alcohol or consists essentially of water. 21.The method of claim 16, further comprising sterilizing the medium afteradding said MSM.
 22. The method of claim 16, wherein said medium is freefrom preservatives.
 23. The method of claim 16, wherein the MSM inhibitsthe microbial activity by reducing growth rate of H1N1 influenza by atleast 50% as compared to the growth rate of H1N1 influenza in theabsence of MSM.